AbstractsBiology & Animal Science

Crimean-Congo hemorragic fever virus : studies on molecular pathogenesis and host-cell interactions

by Helen Karlberg

Institution: Karolinska Institute
Year: 2015
Record ID: 1328074
Full text PDF: http://hdl.handle.net/10616/42336


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.…