Functional dissection of the alphavirus capsid protease: sequence requirements for activity

Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M.; Schaefer, Stephan

doi:10.1186/1743-422x-7-327

Cited by 24 publications

(25 citation statements)

References 18 publications

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“…SDS-PAGE and CBB analysis indicate that the SAV structural (glycol)proteins were expressed in high amounts and that the capsid protein was autocatalytically released from the structural polyprotein, similar as in wildtype alphavirus infection [17], [47]. Alphavirus capsids are multifunctional proteins that are involved in encapsidation of viral genomic RNA, viral budding and in New-world alphaviruses induce host-cell transcription/translational shut-off [17], [48].…”

Section: Discussionmentioning

confidence: 99%

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

et al. 2011

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Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.

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Section: Discussionmentioning

confidence: 99%

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

et al. 2011

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“…The Chik-Wü virus isolate (Genbank: EU037962) or subclones thereof (GenBank: HM369441) served as templates for PCR amplification of the CHIKV capsid and all subclones [3,48]. To clone pCHIKV-CP-EGFP, full-length CHIKV CP was amplified from our previously described expression plasmid in which the protease cleavage site had been inactivated by exchanging serine 213 to alanine [3].…”

Section: Plasmids Cloning and Mutagenesismentioning

confidence: 99%

“…The arthropode-borne Chikungunya virus (CHIKV) of the genus alphavirus in the Togaviridae family is running rampant along the Western and Northern Rim of the Indian Ocean. Outbreaks occurred on its islands including the Malay Archipelago as well as in several tropical African countries [1][2][3]. Alphaviruses express a capsid protein (CP) as part of a polyprotein which, after autoproteolytic cleavage by its protease activity, forms viral particles and packages the viral genomic RNA [3].…”

Section: Introductionmentioning

confidence: 99%

Chikungunya virus capsid protein contains nuclear import and export signals

Thomas

Rai

John

et al. 2013

Virol J

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BackgroundChikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system.MethodsEGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy.ResultsHere we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin α (Karα) for its nuclear translocation. We also found that the Karα4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex.ConclusionsWe were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.

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“…Most of the structure-function analyses have focused on characterization of the inactive form of CP (C-terminal Trp-bound form) using X-ray crystallographic, cryo-electron microscopy, mutational, and biochemical studies (43)(44)(45). In 2008, the enzymatic characterization of the esterase activity of the truncated form of SFV CP was reported (18).…”

Section: Discussionmentioning

confidence: 99%

trans -Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

Aggarwal

Dhindwal

Kumar

et al. 2014

J Virol

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The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation and nucleocapsid core formation, followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential antialphaviral target to arrest capsid release, maturation, and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and of the crystal structure in its active form. Here, we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and the K m value was found to be 2.63 ؎ 0.62 M while the k cat /K m value was 4.97 ؋ 10 4 M ؊1 min ؊1 . Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81-Å resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identify conformational changes in the active site, the oxyanion hole, and the substrate specificity pocket residues, which could be critical for rational drug design. IMPORTANCEThe alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unap- , the disordered C-terminal residues after His261, and the presence of a water molecule in the oxyanion hole of AVCP⌬2 (AVCP with a deletion of the last two residues at the C terminus) reveal the effect of the C-terminal Trp267 deletion on enzyme structure. New structural data reported in this study along with the fluorogenic assay will be useful in substrate specificity characterization, high-throughput protease inhibitor screening, and structure-based development of antiviral drugs.

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Functional dissection of the alphavirus capsid protease: sequence requirements for activity

Cited by 24 publications

References 18 publications

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Chikungunya virus capsid protein contains nuclear import and export signals

trans -Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

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