Polyprotein Processing as a Determinant for in Vitro Activity of Semliki Forest Virus Replicase
Semliki Forest virus (SFV) is an arthropod-borne alphavirus that induces membrane invaginations (spherules) in host cells. These harbor the viral replication complexes (RC) that synthesize viral RNA. Alphaviruses have four replicase or nonstructural proteins (nsPs), nsP1-4, expressed as polyprotein P1234. An early RC, which synthesizes minus-strand RNA, is formed by the polyprotein P123 and the polymerase nsP4. Further proteolytic cleavage results in a late RC consisting of nsP1-4 and synthesizing plus strands. Here, we show that only the late RCs are highly active in RNA synthesis in vitro. Furthermore, we demonstrate that active RCs can be isolated from both virus-infected cells and cells transfected with the wild-type replicase in combination with a plasmid expressing a template RNA. When an uncleavable polyprotein P123 and polymerase nsP4 were expressed together with a template, high levels of minus-strand RNA were produced in cells, but RCs isolated from these cells were hardly active in vitro. Furthermore, we observed that the uncleavable polyprotein P123 and polymerase nsP4, which have previously been shown to form spherules even in the absence of the template, did not replicate an exogenous template. Consequently, we hypothesize that the replicase proteins were sequestered in spherules and were no longer able to recruit a template.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2017 |
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| Erschienen: |
2017 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:9 |
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| Enthalten in: |
Viruses - 9(2017), 10 vom: 07. Okt. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Pietilä, Maija K [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 23.05.2018 Date Revised 09.12.2020 published: Electronic Citation Status MEDLINE |
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| doi: |
10.3390/v9100292 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM276604083 |
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| 100 | 1 | |a Pietilä, Maija K |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Polyprotein Processing as a Determinant for in Vitro Activity of Semliki Forest Virus Replicase |
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| 500 | |a Date Revised 09.12.2020 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Semliki Forest virus (SFV) is an arthropod-borne alphavirus that induces membrane invaginations (spherules) in host cells. These harbor the viral replication complexes (RC) that synthesize viral RNA. Alphaviruses have four replicase or nonstructural proteins (nsPs), nsP1-4, expressed as polyprotein P1234. An early RC, which synthesizes minus-strand RNA, is formed by the polyprotein P123 and the polymerase nsP4. Further proteolytic cleavage results in a late RC consisting of nsP1-4 and synthesizing plus strands. Here, we show that only the late RCs are highly active in RNA synthesis in vitro. Furthermore, we demonstrate that active RCs can be isolated from both virus-infected cells and cells transfected with the wild-type replicase in combination with a plasmid expressing a template RNA. When an uncleavable polyprotein P123 and polymerase nsP4 were expressed together with a template, high levels of minus-strand RNA were produced in cells, but RCs isolated from these cells were hardly active in vitro. Furthermore, we observed that the uncleavable polyprotein P123 and polymerase nsP4, which have previously been shown to form spherules even in the absence of the template, did not replicate an exogenous template. Consequently, we hypothesize that the replicase proteins were sequestered in spherules and were no longer able to recruit a template | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a RNA synthesis | |
| 650 | 4 | |a Semliki Forest virus | |
| 650 | 4 | |a alphavirus | |
| 650 | 4 | |a in vitro replication | |
| 650 | 4 | |a nonstructural protein | |
| 650 | 4 | |a polymerase | |
| 650 | 4 | |a replication complex | |
| 650 | 7 | |a Polyproteins |2 NLM | |
| 650 | 7 | |a RNA, Viral |2 NLM | |
| 650 | 7 | |a Viral Nonstructural Proteins |2 NLM | |
| 650 | 7 | |a RNA-Dependent RNA Polymerase |2 NLM | |
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| 700 | 1 | |a Albulescu, Irina C |e verfasserin |4 aut | |
| 700 | 1 | |a Hemert, Martijn J van |e verfasserin |4 aut | |
| 700 | 1 | |a Ahola, Tero |e verfasserin |4 aut | |
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| 856 | 4 | 0 | |u http://dx.doi.org/10.3390/v9100292 |3 Volltext |
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