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Pathogenesis of gammaherpesviruses based upon structural data
Publication . Cerqueira, Sofia Isabel Arriaga Mimoso, 1988-; Simas, João Pedro, 1965-
The hallmark of herpesviruses is the establishment of lifelong latent infections in specific cell types. Gammaherpesviruses, a herpesvirus subfamily with causative roles in several malignancies, drive proliferation of latently infected B cells in germinal centres (GCs) to expand latency and to achieve long-term persistence in memory B cells. During latency, the viral genome is maintained as a non-integrated circular episome within the host cell nucleus, and viral protein expression is highly restricted. To persist in proliferating cells, viral episomes must replicate in step with normal cell division and segregate to newly formed nuclei after mitosis. This process is mediated by gammaherpesvirus episome maintenance proteins. In particular, gamma-2-herpesviruses encode a latency-associated nuclear antigen (LANA), which has been shown to mediate episome persistence and is also known as a cellular transcription modulator, including through E3 ubiquitin-ligase activity. Remarkably, the crystal structure of LANA DNA binding domain has been solved and revealed several structural features that are associated with specific LANA functions. The aim of this thesis was to address the role of particular LANA functions in the context of gammaherpesvirus latent infection in vivo, using infection of laboratory mouse with murid herpesvirus-4 (MuHV-4) as an experimental model. To this end, MuHV-4 recombinant viruses harbouring structure-based mutations targeting specific MuHV-4 LANA (mLANA) interfaces were engineered, and the ability of these recombinants to establish and maintain latent infection was analysed upon intranasal infection of mice. These experiments demonstrated that mLANA binding to terminal repeat (TR) elements in viral DNA is essential for latency expansion in GC B cells and persistence in the host. Interestingly, mLANA was capable of mediating episome persistence despite mutations that resulted in considerably reduced TR DNA binding, depending on TR elements number present in episomes. This work also showed that the novel mLANA dorsal positive patch, opposite to the DNA binding interface, is required for efficient latency expansion in GC B cells. In addition, recombinants bearing mutations in mLANA suppressor of cytokine signalling (SOCS)-box, which lies within a loop protruding perpendicular to the DNA binding interface, exhibited an attenuation of latency levels in spleen, demonstrating that mLANA E3 ubiquitin-ligase activity contributes to latency amplification. Overall, these findings validate LANA as an essential player in gammaherpesvirus latent infection and reveal the advantage of performing pathogenesis studies guided by structural data. Hence, this study constitutes a primer for pharmacological inhibition of LANA functions, through targeting of specific structural features, as a putative strategy to control gammaherpesvirus latent infection and associated pathologies.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/80152/2011