Crimean-Congo hemorragic fever virus : studies on molecular pathogenesis and host-cell interactions
Author: Karlberg, Helen
Date: 2015-02-04
Location: Samuelssonsalen, Tomtebodavägen 6, Karolinska Institutet, Campus Solna
Time: 09.30
Department: Inst för mikrobiologi, tumör- och cellbiologi / Dept of Microbiology, Tumor and Cell Biology
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Thesis (1.141Mb)
Abstract
Crimean-Congo haemorrhagic fever (CCHF) disease in humans shows a spectrum of severity, from mild to acute. The pathogenesis of this and other viral haemorrhagic fevers (VHF) is associated with alteration of vascular barrier function and haemorrhage, but the specific underlying mechanism is unknown. This thesis examines the progression of the disease, in particular virus-host cell interactions and how CCHFV modulates the function of both Type I IFN response and apoptotic pathways. Programmed cell death and regulation of apoptosis in response to viral infection is an important factor for host or virus survival. In order to establish viral infection and to keep the virus contagious, it is also important to evade antiviral responses such as Type I IFN response.
An initial study showed that replicating CCHFV delays the Type I IFN response, possibly by interfering with the activation pathway of IRF-3. A second study revealed that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, it was demonstrated that this motif is subject to caspase cleavage. It was also demonstrated that CCHFV nucleocapsid (N) protein is cleaved into a 30-kDa fragment while caspase activity is induced during infection and that CCHFV infection induces caspase-3-dependent apoptosis at late post infection. Using caspase inhibitors and cells lacking caspase-3, it was shown that the cleavage of N protein is caspase-3-dependent. The inhibition of apoptosis induced progeny viral titres.
A study examining the crystal structures of CCHFV N revealed two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit and the base of the globular body of the adjacent subunit stabilises the helical organisation of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. Incubation with primer-length ssRNAs revealed the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change in CCHFV N upon deoligomerisation results in exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. These structural findings extend current knowledge regarding CCHFV N structure. Based on the high degree of structural similarity between CCHFV and LASV N proteins, they may have an ancestor in common.
A final study showed that CCHFV has strategies for interplaying with apoptosis pathways, thereby regulating the caspase cascade. There were indications that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, the host cellular response at late post infection appears to induce host cellular pro-apoptotic molecules through the death receptor pathway. External host-derived stimuli probably initiate the apoptotic process and the route continues either by crosstalk between the death receptor and mitochondria routes, or separately.
An initial study showed that replicating CCHFV delays the Type I IFN response, possibly by interfering with the activation pathway of IRF-3. A second study revealed that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, it was demonstrated that this motif is subject to caspase cleavage. It was also demonstrated that CCHFV nucleocapsid (N) protein is cleaved into a 30-kDa fragment while caspase activity is induced during infection and that CCHFV infection induces caspase-3-dependent apoptosis at late post infection. Using caspase inhibitors and cells lacking caspase-3, it was shown that the cleavage of N protein is caspase-3-dependent. The inhibition of apoptosis induced progeny viral titres.
A study examining the crystal structures of CCHFV N revealed two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit and the base of the globular body of the adjacent subunit stabilises the helical organisation of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. Incubation with primer-length ssRNAs revealed the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change in CCHFV N upon deoligomerisation results in exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. These structural findings extend current knowledge regarding CCHFV N structure. Based on the high degree of structural similarity between CCHFV and LASV N proteins, they may have an ancestor in common.
A final study showed that CCHFV has strategies for interplaying with apoptosis pathways, thereby regulating the caspase cascade. There were indications that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, the host cellular response at late post infection appears to induce host cellular pro-apoptotic molecules through the death receptor pathway. External host-derived stimuli probably initiate the apoptotic process and the route continues either by crosstalk between the death receptor and mitochondria routes, or separately.
List of papers:
I. Andersson I, Karlberg H, Mousavi-Jazi M, Martínez-Sobrido L, Weber F, Mirazimi A. Crimean-Congo hemorrhagic fever virus delays activation of the innate immune response. J Med Virol. 2008Aug;80(8):1397-404.
Fulltext (DOI)
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II. Karlberg H, Tan YJ, Mirazimi A. Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection. J Biol Chem. 2011 Feb 4;286(5):3227-34.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Wang Y, Dutta S, Karlberg H, Devignot S, Weber F, Hao Q, Tan YJ, Mirazimi A, Kotaka M. Structure of Crimean-Congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage. J Virol. 2012 Nov; 86 (22):12294-303.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Karlberg H, Tan YJ, Mirazimi A. Crimean-Congo hemorrhagic fever replication interplays with regulation mechanisms of apoptosis. J Gen Virol. [Accepted]
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Andersson I, Karlberg H, Mousavi-Jazi M, Martínez-Sobrido L, Weber F, Mirazimi A. Crimean-Congo hemorrhagic fever virus delays activation of the innate immune response. J Med Virol. 2008Aug;80(8):1397-404.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Karlberg H, Tan YJ, Mirazimi A. Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection. J Biol Chem. 2011 Feb 4;286(5):3227-34.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Wang Y, Dutta S, Karlberg H, Devignot S, Weber F, Hao Q, Tan YJ, Mirazimi A, Kotaka M. Structure of Crimean-Congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage. J Virol. 2012 Nov; 86 (22):12294-303.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Karlberg H, Tan YJ, Mirazimi A. Crimean-Congo hemorrhagic fever replication interplays with regulation mechanisms of apoptosis. J Gen Virol. [Accepted]
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Mirazimi, Ali
Issue date: 2015-01-12
Rights:
Publication year: 2015
ISBN: 978-91-7549-811-9
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