Group outing on the 15th and 16th of April
For our April group seminar, we decided to get out of the lab and surround
ourselves with fresh Mulgimaa air at Taagepera castle. Our program was
stuffed with everything alpha-, flavi- and coronavirus related which has in
some way or other been subject to our research.
We are thankful to our hosts in Taagepera castle for the great accomodation
and good food! We are also glad that our visiting researchers from Brazil and
China could join us on the occasion!
Hopefully we can do this again soon enough!
Sainan Wang succesfully defended her PhD thesis
On the 19th of February 2024, Sainan received her PhD degree
in the Institute of Technology. The work was completed under
professor Merits' supervision and we thank the opponent,
associate professor Kenneth A. Stapleford, for his thorough
work and for the interesting seminar talk.
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The thesis titled "Structure-guided insights into the functions of CHIKV nsP2" summarized in the author's own words:
Alphaviruses are enveloped viruses with positive-strand RNA genomes that are responsible for many human and animal diseases. One of the most medically important alphaviruses is chikungunya virus (CHIKV), which can cause chikungunya fever, an acute febrile disease typically accompanied by rash and debilitating and chronic arthralgia that can last for months or even years. Since its first massive outbreak at 2004, CHIKV has caused millions of cases in over 50 countries and has become a re-emerging pathogen of global importance. However, there are no licensed drugs to treat the disease caused by CHIKV infection. Alphavirus genomic RNA replication depends on the process of RNA synthesis and modification carried out by non-structural proteins (nsPs) encoded by viral RNA. nsP2 is considered as the main driving force and master-regulator of RNA replication. However, the coordination of multiple enzymatic activities and other functions of nsP2 was poorly understood. The current study provided structural basis for better understanding of the roles of nsP2 in the alphavirus replication process; it also indicated that nsP2 can be engineered to perform additional functions required for virus replication. The main conclusions of this study could be illustrated as follows: 1: We identified the crucial residues that formed the stacking interactions between nsP2 and the RNA molecule corresponding to conserved 3’ end of CHIKV genome. Disruption of these interactions was found to have detrimental effect on viral RNA replication. 2: The flexible interdomain linker, connecting the N-terminal helicase and C-terminal protease regions of nsP2, was found to be essential for CHIKV replication as well; only small changes such as the deletion of one amino acid residue and the insertion of up to 10 amino acids residues were tolerated without significant impact on viral replication. However, the deletion of three or five residues of linker was lethal for CHIKV. 3: The insertion of G3BP/Rin binding motifs into the flexible regions of nsP2 (in the linker region or between different domains of nsP2 protease) was not only tolerated but the inserted sequence was able to support replication of mutant CHIKV in both mammalian and insect cells. The same insertion could also restore the infectivity of viruses lacking G3BP binding motifs in nsP3 in mammalian cells.
Laura Sandra Lello succesfully defended her PhD thesis
On the 25th of August, Laura defended her thesis titled "Unraveling the
intricate nature of the alphavirus RNA replicase". The work was supervised
by professor Andres Merits and we thank the opponent professor
Scott Weaver from the University of Texas Medical Branch (USA).
The work summarized in the author's own words:
Alphaviruses are a genus of viruses with a positive-strand RNA genome and include multiple important human pathogens. Alphaviruses that infect mammals, birds and reptiles can be largely divided into two groups. First, viruses that cause illness characterized by arthralgia and second, viruses that cause neurological diseases. One of the most important alphaviruses medically is chikungunya virus, which can be characterized in humans by symptoms like fever, rash and joint pain with the possibility of persisting into months- or even years-long arthritis. The central part in the life cycle of every virus with an RNA genome is the RNA replication. Thus, the RNA replication, i.e., the propagation of the RNA genome, is one of the main targets for the generation of vaccines and antivirals. Despite the wide distribution of alphaviruses among humans, no licenced vaccines or antiviral drugs are available. This emphasizes the importance of acquiring detailed information on the fundamental principles of the RNA replication. The replication of alphavirus genomes is performed by four specific proteins, called the replicase proteins. These replicase proteins assemble into functional replication complexes that carry out the propagation of the RNA genome. In this thesis, the formation of the replication complexes was studied on the example of 10 different alphaviruses. The prerequisite for the formation of active replication complexes is the replicase proteins recognizing their genome, binding it and only then they can initiate the replication. Here, we discovered that some alphavirus replicases can recognize and replicate RNA sequences originating from different alphaviruses, whereas some replicases demonstrated high specificity toward their own genome. Furthermore, we found that functional replication complexes can be formed from replicase proteins which originate from different viruses (i.e., hybrid replicases). Using these hybrid replicases we demonstrated which replicase proteins account for recognizing the virus genome specific elements and determine whether to replicate the genome or not. In addition, we showed that template constructs which include alphavirus genome specific elements and encode for reporter proteins are activated in cells that are infected with relevant alphaviruses. Thus, such biosensors are expressed in cells upon alphavirus infection and the infection of cells can be confirmed by visually observing the infected cells turning green. Finally, using the information obtained in this thesis, we participated in a study which resulted in acquiring the 3D structure of the central part of the alphavirus replicase, the catalytic subunit of the nsP4, and studied the importance of some structurally key elements using structure guided mutagenesis.
Studies included in this thesis offer new information on the RNA replication of alphaviruses. The possibility of forming active hybrid replication complexes sheds light on alphavirus replication in cases where multiple viruses have infected a single cell. The ability of the alphavirus replicases to recognize RNA elements of different viruses offers insights into the possible recombination of the alphavirus genomes and, more generally, the evolution of alphaviruses.
Kai Rausalu succesfully defended her PhD thesis
Kai Rausalu defended her PhD thesis on the 15th of July, 2023
in the Institute of Technology. The work titled "Alphaviral nsP2
protease: requirements for functionality to inhibition" was
supervised by professor Andres Merits and the defense
opponent was professor Margaret Kielian from Albert Einstein
College of Medicine (USA).
The work summarized in the author's own words:
Alphaviruses (genus Alphavirus, family Togaviridae) have an RNA genome and the virions are approximately 70 nm in size. One of alphaviruses is Chikungunya virus (CHIKV), which causes a disease with debilitating joint pains that can last for months. It is important to find antiviral compounds to alleviate suffering caused by CHIKV. For that, we need to understand how the viral life cycle occurs in the cells. nsP2 is a multifunctional CHIKV protein, it is the viral protease and the RNA helicase, nsP2 also participates in the suppression of cellular immune response. As a protease, nsP2 is the viral protein, that cleaves the viral nonstructural polyprotein into individual proteins. This process plays a central role in the regulation of the viral life cycle. Within this thesis, we studied the requirements of CHIKV nsP2 for its protease activity. We found, that CHIKV nsP2 is a cysteine-protease, which is similar to previously studied alphaviral nsP2 proteases. In amino acid sequence CHIKV nsP2 must have intact cysteine 478 for its protease activity. If this cysteine or nearby tryptophan 479 is substituted with alanine, then CHIKV nsP2 loses its protease activity. Cleavage of alphaviral nonstructural polyprotein is a temporally regulated process. We studied how Semliki Forest virus (SFV) and Sindbis virus (SINV) are affected by changes in the speed of cleavages of the nonstructural polyprotein. We found, that it is important for SFV and SINV to prevent premature cleavage between viral nsP1 and nsP2. Accelerated processing of SFV or SINV nonstructural polyprotein caused decrease in infectivities and reduced viral RNA synthesis. The final part of the thesis is the analysis of novel CHIKV inhibitors. The structure of a previously known CHIKV inhibitor was modified in several ways and the anti-CHIKV properties of the novel compounds were evaluated. We identified a compound, that had approximately 10-fold increased anti-CHIKV activity in cell culture.
Liubov Cherkashchenko succesfully defended her PhD thesis
Liubov defended her thesis "New insights into alphaviral
nsP2 functions" on the 10th of February in the Institute of
Technology. The project was supervised by professor
Andres Merits and the opponent on the defense was
associate professor Jelke Fros from Wageningen University,
Netherlands.
The summary of the work in the author's own words:
Over the last decades, the intensive studies of
factors/activities responsible for multiple aspects of
alphavirus infection have been performed. In particular,
our understanding about structures and functions of viral RNA replicase and its components has significantly increased. Novel findings emphasize that one of the first and most essential event in alphavirus infection is processing of the ns polyprotein carried out by its nsP2 region and an individual nsP2; this process not only ensures the release of functional replicase subunits but also determines would these proteins form the active RCs or not. Thus, it is increasingly evident that nsP2 is one of the “main driving forces” of successful RNA replication. Furthermore, due to its versatile functions and various activities nsP2 is involved in other aspects of infection. It is one of the key determinants associated with activation as well as counteracting of antiviral response in infected cells. Therefore, different modifications of the protein, including point mutations, often have drastic impact on alphavirus infection. However, much of the precise mechanisms of P2 action remain enigmatic. What is clear is that nsP2 does not act alone, its activities are modulated by other components of viral replicase.
The current study allowed us to identify and confirm new functions and properties of nsP2 and alphavirus RNA replicase. The general conclusions of this study can be presented as follows:
1. Alphavirus trans-replicase systems can be applied as a tool for studies of functions of nsP2 associated with inhibition of RC formation/activity. These functions are essential for SIE and are likely related to these used to regulate RNA replication in the alphavirus infected cells. Using trans-replicase systems of CHIKV and SINV, it was found that the key event in SIE is targeting of replicase precursor (P1234) by an individual nsP2 protein and that this ability of nsP2 can be altered by mutations present in its functionally important regions.
2. It was found that synthesis of an individual (free) nsP2 in mosquito cells has an inhibitory effect on the alphavirus RC formation/functionality. This is not, however, a result of a single mechanism but results from combination of nsP2 protease-activity dependent mechanism and protease-activity independent mechanisms. The level and dominant mode of inhibition of alphavirus RNA replication depends form the virus, source of free nsP2 and substitutions present in this protein. The protease-activity mediated mechanism is important for suppression of replication of matching virus and relies mostly on ability of nsP2 to cleave 2/3 site in ns polyprotein. For some viruses such as SINV it is also dominant mechanism used to suppress formation of RNA replicases of heterologous alphaviruses.
3. nsP2 of SINV and CHIKV can inhibit formation/activity of RNA replicase of heterologous alphavirus using protease-activity independent mechanisms; for nsP2 of CHIKV this is the dominant mechanism to suppress activity of RNA replicases of heterologous alphaviruses. The precise details of protease-activity independent mode of action of nsP2 remain unknown; however, it is clear that this property can be enhanced by introduction of certain mutations into nsP2. It is likely that the protease-activity independent inhibitory effect originates not from a single mechanism but from several mechanisms.
4. Trans-replicase of highly pathogenic EEEV was found to be highly active in human and mosquito cells. Splitting of construct of EEEV P1234 expression into two (P123 and nsP4) or three (nsP1, P23 and nsP4) expression construct allowed analysis of requirements of active RC formation. It was found that activity of EEEV RNA replicase depends from correct ratio of P123 (or nsP1+P23) component to nsP4 component and that in contrast to previously studied alphaviruses an excess of nsP4 reduced activity of EEEV RNA replicase. The reduction was due to the decrease of a number of cells were RNA replication was initiated as well as to the reduced RNA replicase activity in such cells. It remains unclear, is this property unique for EEEV RNA replicase.
5. Natural isolate RRV 2528 was found to be a prominent inducer of type-I IFN expression. This property was associated with specific amino acid substitutions in the nsP2 encoded by this isolate. None of these substitutions or their combination affected ability of RRV to induce shutdown of cellular protein synthesis or level of viral structural proteins expression. Similarly, no effect on the processing of P1234 was detected. Combined, these findings indicate that the excessive type-I IFN induction was not due to the lack of the ability to induce general shutdown of cellular gene expression or due to the defects in RC formation.
