Vaccinia Virus Entry Is Followed by Core Activation and Proteasome-Mediated Release of the Immunomodulatory Effector VH1 from Lateral Bodies

Schmidt, Florian I.; Bleck, Christopher K. E.; Reh, Lucia; Novy, Karel; Wollscheid, Bernd; Helenius, Ari; Stahlberg, Henning; Mercer, Jason

doi:10.1016/j.celrep.2013.06.028

Cited by 90 publications

(137 citation statements)

References 87 publications

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“…WR EGFP‐A4 MVs were first allowed to bind to cells on ice in the absence or presence of YM201636. When cell‐associated fluorescence was quantified by flow cytometry as described 55, no defect in virus binding to cells was observed (Figure 4D).…”

Section: Resultsmentioning

confidence: 98%

“…We have previously employed this strategy to test the specificity of entry inhibitors 34, 55. To determine whether YM201636 inhibited MV entry, WR E EGFP MVs were bound to cells in the presence or absence of YM201636 and subsequently treated with pH 5.0 media (Figure 4E).…”

Section: Resultsmentioning

confidence: 99%

“…EVs consist of an MV‐like particle surrounded by an additional Golgi‐derived membrane containing cellular and at least six additional viral proteins 56. We have previously demonstrated that similar to MVs, EVs enter HeLa cells by inducing their own macropinocytic uptake 55. To overcome the topological constraint of having two membranes, the outer EV membrane is shed in an acid‐dependent fashion within macropinosomes to free the underlying MV for fusion 36.…”

Section: Resultsmentioning

confidence: 99%

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Vaccinia Virus Infection Requires Maturation of Macropinosomes

Rizopoulos

Balistreri

Kilcher

et al. 2015

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The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live‐cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome‐associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow‐up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late‐penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Vaccinia Virus Infection Requires Maturation of Macropinosomes

Rizopoulos

Balistreri

Kilcher

et al. 2015

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“…Thin sections of virus-infected cells were probed with antibodies against the A3 (4b), A4 (p39), F17 (VP11), and L4 proteins. A3 and A4 are thought to be core wall proteins, and F17 is thought to be a lateral body protein (33)(34)(35)(36). Figure 1 shows that antibodies against L4 decorate the center of the core, distinct from A3, A4, and F17, which have a more peripheral localization.…”

Section: Resultsmentioning

confidence: 99%

Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

Jesus

Moussatché

Condit

2014

J Virol

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Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCEThe vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid.

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“…This protein induces mechanical ruptures at the endosomal membrane to allow virus endosomal escape to the cytosol (59). Like other enveloped viruses, poxviruses respond to uncoating cues immediately after fusion, and viral cores undergo dramatic morphological changes (60). Vaccinia virus genome uncoating follows a multistep process.…”

Section: Discussionmentioning

confidence: 99%

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

Cuesta-Geijo

Chiappi

Galindo

et al. 2016

J Virol

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African swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection. IMPORTANCESince its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV.

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Vaccinia Virus Entry Is Followed by Core Activation and Proteasome-Mediated Release of the Immunomodulatory Effector VH1 from Lateral Bodies

Cited by 90 publications

References 87 publications

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

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