Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag Assembly
Our previous studies identified the 1,4,5-inositol triphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initi...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2014-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00234/full |
| _version_ | 1818877858651570176 |
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| author | Lorna S Ehrlich Sara ePhotiadis Gisselle N Medina Paul B Whittredge Justin W Taraska Susan eWatanabe Carol A Carter |
| author_facet | Lorna S Ehrlich Sara ePhotiadis Gisselle N Medina Paul B Whittredge Justin W Taraska Susan eWatanabe Carol A Carter |
| author_sort | Lorna S Ehrlich |
| collection | DOAJ |
| description | Our previous studies identified the 1,4,5-inositol triphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca2+ promotes viral particle production and our studies indicate that IP3R and the ER Ca2+ store are the physiological providers of Ca2+ for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca2+ level originating from the ER store than control cells, suggesting that Gag induced release of store Ca2+. This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca2+ elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence and immunoelectron microscopy or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an ability to mod |
| first_indexed | 2024-12-19T14:04:58Z |
| format | Article |
| id | doaj.art-fd2ae4376fcb413989f95ea6ac7a5593 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-19T14:04:58Z |
| publishDate | 2014-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-fd2ae4376fcb413989f95ea6ac7a55932022-12-21T20:18:20ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2014-05-01510.3389/fmicb.2014.0023479739Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag AssemblyLorna S Ehrlich0Sara ePhotiadis1Gisselle N Medina2Paul B Whittredge3Justin W Taraska4Susan eWatanabe5Carol A Carter6Stony Brook UniversityStony Brook UniversityStony Brook UniversityNational Heart Lung and Blood Institute NIHNational Heart Lung and Blood Institute NIHStony Brook UniversityStony Brook UniversityOur previous studies identified the 1,4,5-inositol triphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca2+ promotes viral particle production and our studies indicate that IP3R and the ER Ca2+ store are the physiological providers of Ca2+ for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca2+ level originating from the ER store than control cells, suggesting that Gag induced release of store Ca2+. This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca2+ elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence and immunoelectron microscopy or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an ability to modhttp://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00234/fullHIV-1Ca2+ signalingIP3RGag assemblyL DomainTsg101 |
| spellingShingle | Lorna S Ehrlich Sara ePhotiadis Gisselle N Medina Paul B Whittredge Justin W Taraska Susan eWatanabe Carol A Carter Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag Assembly Frontiers in Microbiology HIV-1 Ca2+ signaling IP3R Gag assembly L Domain Tsg101 |
| title | Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag Assembly |
| title_full | Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag Assembly |
| title_fullStr | Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag Assembly |
| title_full_unstemmed | Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag Assembly |
| title_short | Tsg101 Regulates PI(4,5)P2/Ca2+ Signaling for HIV-1 Gag Assembly |
| title_sort | tsg101 regulates pi 4 5 p2 ca2 signaling for hiv 1 gag assembly |
| topic | HIV-1 Ca2+ signaling IP3R Gag assembly L Domain Tsg101 |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00234/full |
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