Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication

Soares, Jamária A. P.; Leite, Flávia G. G.; Andrade, Luciana Garcia; Torres, Alice A.; Sousa, Lirlândia P.; Barcelos, Lucíola da Silva; Teixeira, Mauro M.; Ferreira, Paulo César Peregrino; Kroon, Erna Geessien; Souto-Padrón, Thaïs; Bonjardim, Cláudio Antônio

doi:10.1128/jvi.00245-09

Cited by 116 publications

(118 citation statements)

References 94 publications

(143 reference statements)

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“…Our RNAi screen indicated that PI(3)K, PI(4)K, PI(5)K and PIKfyve were needed for MV infection (Figure 2). Previous studies have demonstrated a role for PI(3)K during multiple stages of VACV infection 68, 69 including entry 34, 39, 70. Consistent with this, PtdIns(3)P was detected on early MV‐containing macropinosomes until 30 min suggesting that these compartments undergo a lipid switch during their maturation (Figure 4A,B).…”

Section: Discussionsupporting

confidence: 82%

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: Discussionsupporting

confidence: 82%

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Rizopoulos

Balistreri

Kilcher

et al. 2015

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“…GM-CSF receptor activation is followed by Jak2 phosphorylation of receptor tyrosines and eventual activation of Stat5 and the MAPK pathways via Grb2, Shc, and SHP2 (66). Similarly, VACV is reported to be dependent on MAPK signaling for replication (45,46,47). We observed significantly decreased viral production with treatment of Akt and Erk inhibitors (Fig.…”

Section: Discussionsupporting

confidence: 47%

“…VACV is dependent on Akt and Erk1/2 signaling pathways for entry and replication (45)(46)(47)(48). Since we have discovered that M1 and M2 cells are permissive to VACV, we sought to analyze the dependence of these two pathways on VACV replication in MDMs independent of binding and entry.…”

Section: Resultsmentioning

confidence: 99%

Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination

Byrd

Shepherd

Lan

et al. 2014

J Virol

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Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serumderived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-␥) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1␤ treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. O rthopoxvirus, a genus of the subfamily Chordopoxvirinae of the family Poxviridae, is composed of large DNA viruses that infect a wide array of mammalian species. The most famous member of the genus is the human-specific variola virus, which causes smallpox. In nature, variola virus has a strict human-specific tropism, and nonhuman reservoirs of the virus have never been found. Variola virus is generally transmitted via inhalation, with subsequent infection and replication in epithelial cells of the oral and respiratory mucosa. The next stage of infection involves viral infiltration of lymphoid organs accompanied by strong viremia and skin lesions. Recent studies using high doses of variola virus to infect Cynomolgus macaques in an attempt to develop an animal model of smallpox have demonstrated that infected animals develop systemic infection and hemorrhagic symptoms (1, 2). These symptoms were correlated with monocyte/macrophage-mediated viremia and dissemination (1, 2). In mice, macrophages are crucial to control the infection of the orthopoxvirus ectromelia virus (ECTV) (3, 4). However, ECTV replicates in macrophages (5) and directly contributes to dissemination within the host (6). Giv...

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“…Vaccinia virus is known to manipulate hostsignalling pathways, such as MAPK (3) and Akt (38,39) pathways, to enhance viral replication. Using a kinase array, we showed that infection with GLV-1h68 activated JNK pathways in both V600E BRAF mutant and BRAF/RAS wild-type cells.…”

Section: Discussionmentioning

confidence: 99%

Synergistic cytotoxicity of radiation and oncolytic Lister strain vaccinia in V600D/EBRAF mutant melanoma depends on JNK and TNF-α signaling

Khan

Mansfield

et al. 2013

Oncogene

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Melanoma is an aggressive skin cancer that carries an extremely poor prognosis when local invasion, nodal spread or systemic metastasis has occurred. Recent advances in melanoma biology have revealed that RAS-RAF-MEK-ERK signalling plays a pivotal role in governing disease progression and treatment resistance. Proof-of-concept clinical studies have shown that direct BRAF inhibition yields impressive responses in advanced disease but these are short-lived as treatment resistance rapidly emerges. Therefore, there is a pressing need to develop new targeted strategies for BRAF mutant melanoma.As such, oncolytic viruses represent a promising cancer-specific approach with significant activity in melanoma.This study investigated interactions between genetically-modified vaccinia virus (GLV1h68) and radiotherapy in melanoma cell lines with BRAF mutant, Ras mutant or wildtype genotype. Pre-clinical studies revealed that GLV-1h68 combined with radiotherapy significantly increased cytotoxicity and apoptosis relative to either single agent in V600D BRAF/ V600E BRAF mutant melanoma in vitro and in vivo. The mechanism of enhanced cytotoxicity with GLV-1h68/radiation was independent of viral replication and due to attenuation of JNK, p38 and ERK MAPK phosphorylation specifically in BRAF mutant cells. Further studies showed that JNK pathway inhibition sensitized BRAF mutant cells to GLV-1h68-mediated cell death, mimicking the effect of radiation. GLV1h68 infection activated MAPK signalling in V600D BRAF/ V600E BRAF mutant cell lines and this was associated with TNF-α secretion which, in turn, provided a prosurvival signal.Combination GLV-1h68/radiation (or GLV-1h68/JNK inhibition) caused abrogation of TNF-α secretion. These data provide a strong rationale for combining GLV-1h68 with irradiation in V600D/E BRAF mutant tumors.3

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Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication

Cited by 116 publications

References 94 publications

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination

Synergistic cytotoxicity of radiation and oncolytic Lister strain vaccinia in V600D/EBRAF mutant melanoma depends on JNK and TNF-α signaling

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