Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease.

Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1-/- mice results in replication of HSV-1 and Asah1-/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.

Multivesicular body sorting and the exosomal pathway are required for the release of rat hepatitis E virus from infected cells.

Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.

Turnip Mosaic Virus Components Are Released into the Extracellular Space by Vesicles in Infected Leaves.

Turnip mosaic virus (TuMV) reorganizes the endomembrane system of the infected cell to generate endoplasmic-reticulum-derived motile vesicles containing viral replication complexes. The membrane-associated viral protein 6K2 plays a key role in the formation of these vesicles. Using confocal microscopy, we observed that this viral protein, a marker for viral replication complexes, localized in the extracellular space of infected Nicotiana benthamiana leaves. Previously, we showed that viral RNA is associated with multivesicular bodies (MVBs). Here, using transmission electron microscopy, we observed the proliferation of MVBs during infection and their fusion with the plasma membrane that resulted in the release of their intraluminal vesicles in the extracellular space. Immunogold labeling with a monoclonal antibody that recognizes double-stranded RNA indicated that the released vesicles contained viral RNA. Focused ion beam-extreme high-resolution scanning electron microscopy was used to generate a three-dimensional image that showed extracellular vesicles in the cell wall. The presence of TuMV proteins in the extracellular space was confirmed by proteomic analysis of purified extracellular vesicles from N benthamiana and Arabidopsis (Arabidopsis thaliana). Host proteins involved in biotic defense and in interorganelle vesicular exchange were also detected. The association of extracellular vesicles with viral proteins and RNA emphasizes the implication of the plant extracellular space in viral infection.

The regulation of Endosomal Sorting Complex Required for Transport and accessory proteins in multivesicular body sorting and enveloped viral budding - An overview.

ESCRT (Endosomal Sorting Complex Required for Transport) machinery drives different cellular processes such as endosomal sorting, organelle biogenesis, vesicular trafficking, maintenance of plasma membrane integrity, membrane fission during cytokinesis and enveloped virus budding. The normal cycle of assembly and disassembly of some ESCRT complexes at the membrane requires the AAA-ATPase vacuolar protein sorting 4 (Vps4p). A number of ESCRT proteins are hijacked by clinically significant enveloped viruses including Ebola, and Human Immunodeficiency Virus (HIV) to enable enveloped virus budding and Vps4p provides energy for the disassembly/recycling of these ESCRT proteins. Several years ago, the failure of the terminal budding process of HIV following Vps4 protein inhibition was published; although at that time a detailed understanding of the molecular players was missing. However, later it was acknowledged that the ESCRT machinery has a role in enveloped virus budding from cells due to its role in the multivesicular body (MVB) sorting pathway. The MVB sorting pathway facilitates several cellular activities in uninfected cells, such as the down-regulation of signaling through cell surface receptors as well as the process of viral budding from infected host cells. In this review, we focus on summarising the functional organisation of ESCRT proteins at the membrane and the role of ESCRT machinery and Vps4p during MVB sorting and enveloped viral budding.

The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus.

Hepatitis C virus (HCV) particles are described as lipoviroparticles which are released similarly to very-low-density lipoproteins (VLDLs). However, the release mechanism is still poorly understood; the canonical endoplasmic reticulum-Golgi intermediate compartment (ERGIC) pathway as well as endosome-dependent release has been proposed. Recently, the role of exosomes in the transmission of HCV has been reported. Only a minor fraction of the de novo-synthesized lipoviroparticles is released by the infected cell. To investigate the relevance of multivesicular bodies (MVBs) for viral morphogenesis and release, the MVB inhibitor U18666A was used. Intracellular trafficking was analyzed by confocal microscopy and electron microscopy. Moreover, an mCherry-tagged HCV variant was used. Conditions were established that enable U18666A-dependent inhibition of MVBs without affecting viral replication. Under these conditions, significant inhibition of the HCV release was observed. The assembly of viral particles is not affected. In U18666A-treated cells, intact infectious viral particles accumulate in CD63-positive exosomal structures and large dysfunctional lysosomal structures (multilamellar bodies). These retained particles possess a lower density, reflecting a misloading with lipids. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway. Endosomes facilitate the sorting of HCV particles for release or degradation. IMPORTANCE: There are still a variety of open questions regarding morphogenesis and release of hepatitis C virus. The HCV-infected cell produces significant more viral particles that are released, raising the question about the fate of the nonreleased particles. Moreover, the relevance of the endosomal pathway for the release of HCV is under debate. Use of the MVB (multivesicular body) inhibitor U18666A enabled a detailed analysis of the impact of MVBs for viral morphogenesis and release. It was revealed that infectious, fully assembled HCV particles are either MVB-dependently released or intracellularly degraded by the lysosome. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway independent from the constitutive secretory pathway. Our study describes a so-far-unprecedented cross talk between two pathways regulating on the one hand the release of infectious viral particles and on the other hand the intracellular degradation of nonreleased particles.

Mutation of Glycosylation Sites in BST-2 Leads to Its Accumulation at Intracellular CD63-Positive Vesicles without Affecting Its Antiviral Activity against Multivesicular Body-Targeted HIV-1 and Hepatitis B Virus.

BST-2/tetherin blocks the release of various enveloped viruses including HIV-1 with a "physical tethering" model. The detailed contribution of N-linked glycosylation to this model is controversial. Here, we confirmed that mutation of glycosylation sites exerted an effect of post-translational mis-trafficking, leading to an accumulation of BST-2 at intracellular CD63-positive vesicles. BST-2 with this phenotype potently inhibited the release of multivesicular body-targeted HIV-1 and hepatitis B virus, without affecting the co-localization of BST-2 with EEA1 and LAMP1. These results suggest that N-linked glycosylation of human BST-2 is dispensable for intracellular virion retention and imply that this recently discovered intracellular tethering function may be evolutionarily distinguished from the canonical antiviral function of BST-2 by tethering nascent virions at the cell surface.

Ebola virus host cell entry.

Ebola virus is an enveloped virus with filamentous structure and causes a severe hemorrhagic fever in human and nonhuman primates. Host cell entry is the first essential step in the viral life cycle, which has been extensively studied as one of the therapeutic targets. A virus factor of cell entry is a surface glycoprotein (GP), which is an only essential viral protein in the step, as well as the unique particle structure. The virus also interacts with a lot of host factors to successfully enter host cells. Ebola virus at first binds to cell surface proteins and internalizes into cells, followed by trafficking through endosomal vesicles to intracellular acidic compartments. There, host proteases process GPs, which can interact with an intracellular receptor. Then, under an appropriate circumstance, viral and endosomal membranes are fused, which is enhanced by major structural changes of GPs, to complete host cell entry. Recently the basic research of Ebola virus infection mechanism has markedly progressed, largely contributed by identification of host factors and detailed structural analyses of GPs. This article highlights the mechanism of Ebola virus host cell entry, including recent findings.

Suppression of production of baboon endogenous virus by dominant negative mutants of cellular factors involved in multivesicular body sorting pathway.

Baboon endogenous virus (BaEV) is an infectious endogenous gammaretrovirus isolated from a baboon placenta. BaEV-related sequences have been identified in both Old World monkeys and African apes, but not in humans or Asian apes. Recently, it was reported that BaEV-like particles were produced from Vero cells derived from African green monkeys by chemical induction, and thus BaEV-like particles may contaminate biological products manufactured using Vero cells. In this study, we constructed an infectious molecular clone of BaEV strain M7. We found two putative L-domain motifs, PPPY and PSAP, in the pp15 region of Gag. To examine the function of the L-domain motifs, we conducted virus budding assay using L-domain motif mutants. We revealed that the PPPY motif, but not the PSAP motif, plays a major role as the L-domain in BaEV budding. We also demonstrated that Vps4A/B are involved in BaEV budding. These data suggest that BaEV Gag recruits the cellular endosomal sorting complex required for transport (ESCRT) machinery through the interaction of the PPPY L-domain with cellular factors. These data will be useful for controlling contamination of BaEV-like particles in biological products in the future.

Permeability changes of integrin-containing multivesicular structures triggered by picornavirus entry.

Cellular uptake of clustered α2ß1-integrin induces the formation of membrane compartments that subsequently mature into a multivesicular body (MVB). Enhanced internalization mediated by clustered integrins was observed upon infection by the picornavirus echovirus 1 (EVI). We elucidated the structural features of virus-induced MVBs (vMVBs) in comparison to antibody-induced control MVBs (mock infection) by means of high-pressure cryo fixation of cells followed by immuno electron tomography during early entry of the virus. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and the intraluminal vesicles (ILVs) at 2 and 3.5 hours post infection (p.i.), in contrast to the control MVBs without virus. Breakages in the membranes of vMVBs were detected from tomograms after 2 and especially after 3.5 h suggesting that these breakages could facilitate the genome release to the cytoplasm. The in situ neutral-red labeling of viral genome showed that virus uncoating starts as early as 30 min p.i., while an increase of permeability was detected in the vMVBs between 1 and 3 hours p.i., based on a confocal microscopy assay. Altogether, the data show marked morphological changes in size and permeability of the endosomes in the infectious entry pathway of this non-enveloped enterovirus and suggest that the formed breakages facilitate the transfer of the genome to the cytoplasm for replication.

Crimean-Congo hemorrhagic fever virus entry into host cells occurs through the multivesicular body and requires ESCRT regulators.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus causing outbreaks of severe disease in humans, with a fatality rate approaching 30%. There are no widely accepted therapeutics available to prevent or treat the disease. CCHFV enters host cells through clathrin-mediated endocytosis and is subsequently transported to an acidified compartment where the fusion of virus envelope with cellular membranes takes place. To better understand the uptake pathway, we sought to identify host factors controlling CCHFV transport through the cell. We demonstrate that after passing through early endosomes in a Rab5-dependent manner, CCHFV is delivered to multivesicular bodies (MVBs). Virus particles localized to MVBs approximately 1 hour after infection and affected the distribution of the organelle within cells. Interestingly, blocking Rab7 activity had no effect on association of the virus with MVBs. Productive virus infection depended on phosphatidylinositol 3-kinase (PI3K) activity, which meditates the formation of functional MVBs. Silencing Tsg101, Vps24, Vps4B, or Alix/Aip1, components of the endosomal sorting complex required for transport (ESCRT) pathway controlling MVB biogenesis, inhibited infection of wild-type virus as well as a novel pseudotyped vesicular stomatitis virus (VSV) bearing CCHFV glycoprotein, supporting a role for the MVB pathway in CCHFV entry. We further demonstrate that blocking transport out of MVBs still allowed virus entry while preventing vesicular acidification, required for membrane fusion, trapped virions in the MVBs. These findings suggest that MVBs are necessary for infection and are the sites of virus-endosome membrane fusion.

Coxsackievirus A9 infects cells via nonacidic multivesicular bodies.

UNLABELLED: Coxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae. According to earlier studies, CVA9 binds to αVß3 and αVß6 integrins on the cell surface and utilizes ß2-microglobulin, dynamin, and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here, based on electron microscopy, that CVA9 is found in multivesicular structures 2 h postinfection (p.i.). A neutral red labeling assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicular bodies (MVBs) is crucial for promoting infection, as judged by the strong inhibitory effect of the wild-type form of Hrs and dominant negative form of VPS4 in CVA9 infection. CVA9 infection is dependent on phospholipase C at the start of infection, whereas Rac1 is especially important between 1 and 3 h p.i., when the virus is in endosomes. Several lines of evidence implicate that low pH does not play a role in CVA9 infection. The infection is not affected by Bafilomycin A1. In addition, CVA9 is not targeted to acidic late endosomes or lysosomes, and the MVBs accumulating CVA9 have a neutral pH. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger neutral MVBs, which are important for virus infection. IMPORTANCE: We demonstrate here that the enterovirus coxsackievirus A9 (CVA9) uses a nonclathrin and nonacidic pathway to infect cells. CVA9 does not accumulate in conventional late endosomes or lysosomes. We found that inhibitors of phospholipase C (PLC), Rac1, and the Na(+)/H(+) exchanger decreased CVA9 infection. The PLC inhibitor acts on early entry, the Rac1 inhibitor acts between 1 and 3 h, when the virus is in endosomes, and the Na(+)/H(+) exchange inhibitor acts during various steps during the virus life cycle. The infection depends on the formation of novel neutral multivesicular bodies (MVBs), which accumulate CVA9 during the first hours of entry. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger formation of neutral MVBs. The data show that these enteroviruses favor nonacidic conditions and complex MVBs to promote virus infection.

Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies.

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.

TANK-binding kinase 1 attenuates PTAP-dependent retroviral budding through targeting endosomal sorting complex required for transport-I.

Retroviruses need to bud from producer cells to spread infection. To facilitate its budding, some virus hijacks the multivesicular body (MVB) pathway that is normally used to cargo and degrade ubiquitylated cellular proteins, through interaction between the late domain of Gag polyproteins and the components of MVB machinery. In this study, we demonstrated that TANK-binding kinase 1 (TBK1) directly interacted with VPS37C, a subunit of endosomal sorting complex required for transport-I (ESCRT-I) in the MVB pathway, without affecting the ultrastructure or general function of MVB. Interestingly, overexpression of TBK1 attenuated, whereas short hairpin RNA interference of TBK1 enhanced HIV-1 pseudovirus release from Vero cells in type I IFN (IFN-I)-independent manner. Down-regulation of TBK1 by short hairpin RNA in TZM-bl cells also enhanced live HIV-1 NL4-3 or JR-CSF virus budding without involvement of IFN-I induction. Furthermore, infection of TBK1-deficient mouse embryonic fibroblast cells with a chimeric murine leukemia virus/p6, whose PPPY motif was replaced by PTAP motif of HIV-1, showed that lack of TBK1 significantly enhanced PTAP-dependent, but not PPPY-dependent retrovirus budding. Finally, phosphorylation of VPS37C by TBK1 might regulate the viral budding efficiency, because overexpression of the kinase-inactive mutant of TBK1 (TBK1-K38A) in Vero cells accelerated HIV-1 pseudovirus budding. Therefore, through tethering to VPS37C of the ESCRT-I complex, TBK1 controlled the speed of PTAP-dependent retroviral budding through phosphorylation of VPS37C, which would serve as a novel mechanism of host cell defense independent of IFN-I signaling.

New isolates of carnation Italian ringspot virus differ from the original one by having replication-associated proteins with a typical tombusvirus-like N-terminus and by inducing peroxisome- rather than mitochondrion-derived multivesicular bodies.

Five new isolates of carnation Italian ringspot virus (CIRV) from cherry trees, Gypsophila and surface water differ from the original carnation isolate (CIRV-car) and also from Pelargonium necrotic spot virus (PelNSV) by having an ORF 1/ORF1-RT with a typical tombusvirus-like 5'end and by inducing the formation of peroxisome- rather than mitochondrion-derived multivesicular bodies (MVBs). This supports with natural isolates earlier conclusions reached by others with artificially produced hybrid viruses that the 5'end of ORF 1 determines from which organelle the MBVs will be derived. CIRV-car might have resulted from a natural recombination event with genome elements of a PelNSV-like virus.