Supervisor: prof. Andres Merits (Tartu Ülikool)
Opponent: Prof. Marc Lecuit, PhD
Biology of Infection Unit, Institut Pasteur, France
Semliki Forest virus (SFV) belongs to genus Alphavirus, family Togaviridae. There are many important pathogens among alphaviruses and their infection can cause various illnesses to animals and humans. In nature mosquitoes are responsible for the spread of alphaviruses. In addition, alphaviruses are used as tools in biotechnology. Alphaviruses have a genome with positive polarity that is used in infected cells to translate nonstructural polyprotein that will be processed into four subunits (nsP1-nsP4). Nonstructural proteins are needed to form replication complexes.
In the present work it was discovered that one nonstructural protein nsP3 has degradation signal at its carboxyterminal end. However, it overlaps with the sequence element that acts as a recognition element in the polyprotein processing resulting in the release of nsP3 from nsP4.
In addition, it was found that upstream of the degradation signal nsP3 has a sequence motif in two copies. Further analysis showed that at least one of the motifs is required to bind G3BP1 and G3BP2. Those proteins participate in the formation of stress granules; stress granules store cellular RNAs if the conditions are not favourable. However, cells infected with alphaviruses loose that ability, one of the mechanisms behind the phenomenon is recruitment of G3BP1 and G3BP2 to virus replications complexes.
Although we know that G3BP1 and G3BP2 bind SFV replication complexes there is little knowledge about the other cellular proteins that could do the same. SFV replication complex are bound to lysosomes. If the cells are fed with dextran-covered magnetic nanoparticles those particles end up in lysosomes allowing magnetic sorting of those cellular vesicles. By using quantitative proteomics, it was found that 80 proteins were enriched in the lysosomal fraction from infected cells. Four of them (PCBP1, hnRNP M, hnRNP C, hnRNP K) were chosen for further analysis. All four colocalized with SFV replication complexes. By using siRNA-based knockdown, it was found that knockdown of hnRNP M and hnRNP C enhanced virus replication, knockdown of PCBP1 reduced viral RNA translation and trageting of hnRNP K increased viral synhtesis.
Understanding of alphavirus interactions with host is required to develop new ways to treat the disease.