Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

Pathogenic Mechanisms of Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy (PLOSL)

PATHOGENIC MECHANISMS OF PLOSL Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease, is a recessively inherited disease of brain and bone. PLOSL manifests as early-onset progressive dementia and bone fractures. Mutations in the TYROBP (DAP12) and TREM2 genes have been identified as the primary cause of PLOSL. DAP12 and TREM2 encode important signalling molecules in cells of the innate immune system. The mechanism by which loss-of-function of the DAP12/TREM2 signalling complex leads to PLOSL is currently unknown. The aim of this thesis work was to gain insight into the pathogenic mechanisms behind PLOSL. To first identify the central nervous system (CNS) cell types that express both Dap12 and Trem2, the expression patterns of Dap12 and Trem2 in mouse CNS were analyzed. Dap12 and Trem2 expression was seen from embryonic stage to adulthood and microglial cells and oligodendrocytes were identified as the major Dap12/Trem2 producing cells of the CNS. To subsequently identify the pathways and biological processes associated with DAP12/TREM2 mediated signalling in human cells, genome wide transcript analysis of in vitro differentiated dendritic cells (DCs) of PLOSL patients representing functional knockouts of either DAP12 or TREM2 was performed. Both DAP12 and TREM2 deficient cells differentiated into DCs and responded to pathogenic stimuli. However, the DCs showed morphological differences compared to control cells due to defects in the actin filaments. Transcript profiles of the patient DCs showed differential expression of genes involved in immune response and for genes earlier associated with other disorders of the CNS as well as genes involved in the remodeling of bone, linking the findings with the tissue phenotype of PLOSL patients. To analyze the effect of Dap12 deficiency in the CNS, genome wide expression analysis of Dap12 deficient mouse brain and Dap12 deficient microglia as well as functional analysis of Dap12 deficient microglia was performed. Regulation of several pathways involved in synaptic function and transcripts coding for the myelin components was seen in Dap12 knockout mice. Decreased migration, morphological changes and shortened lifespan of the Dap12 knockout microglia was further observed. Taken together, this thesis work showed that both Dap12 and Trem2 are expressed by CNS microglia and that Dap12 deficiency results in functional defects of these cells. Lack of Dap12 in the CNS also leads to synaptic abnormalities even before pathological changes are seen in the tissue level.This work further showed that loss-of-function of DAP12 or TREM2 leads to changes in morphology and gene expression in human dendritic cells. These data underline the functional diversity of the molecules of the innate immune system and implies their significant contribution also in demyelinating CNS disorders, including those resulting in dementia.