Biotehnoloogia magistritööd - Master's theses

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Browsing Biotehnoloogia magistritööd - Master's theses by Subject "alphavirus"

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    Construction and functional analysis of stable cell lines expressing nsP1 of alphaviruses
    (Tartu Ülikool, 2023) Mirzajani Sarvandani, Mandana; Wang, Sainan, juhendaja; Merits, Andres, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Alphaviruses are positive-strand RNA viruses that are transmitted by mosquitoes and cause various (often serious) diseases in their vertebrate hosts, including humans. Similar to other positive-strand RNA viruses, alphaviruses encode for RNA replicase. RNA replicase of alphaviruses consists of four virus-encoded subunits, called non-structural proteins 1-4 (nsP1-4). nsP1 is a membrane anchor or the replicase complex. It forms a dodecameric ring that holds other components in their place, its guanine-7-methyltransferase (MTase) and guanylyltransferase (GTase) activities are essential for the capping of viral RNA genomes. In this study, tetracycline-inducible stable cell lines expressing nsP1 or its mutants of different alphaviruses were generated. Trans- complementation assay was performed by supplementing homologous or heterologous wild-type nsP1 or its mutant version to functionally defective replicase of Chikungunya virus (CHIKV) in which the defect was caused by mutations of nsP1. We found that expression of wild-type nsP1 of matching or closely related alphaviruses can significantly compensate for capping defects of CHIKV replicase. The compensation was less effective, or not observed at all, for replicase mutants where the defect was due to its compromised membrane attachments. Adding the defective nsP1 to the CHIKV replicase which harbours the same mutation worsens the activity of replicase; however, activities of mutated replicase were restored when it was supplemented with nsP1 harbouring different functional defects. Furthermore, expression of these findings expands our knowledge about alphavirus RNA replication and trans-complementation of replicase-associated defects. Besides, tetracycline-inducible stable cell lines can be used as tools to trans-complement alphaviruses with lethal defects in nsP1 allowing obtaining conditionally infectious alphaviruses virions which can be used at low biosafety level laboratories for high-throughput neutralization and antiviral testing.
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    The novel trans-complementation system for chikungunya virus
    (Tartu Ülikool, 2024) Makhotina, Anna; Merits, Andres, juhendaja; Naumenko, Krystyna, juhendaja; Wang, Sainan, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Alphaviruses (Togaviridae) are mosquito transmitted positive-sense RNA viruses that cause severe and sometimes even lethal diseases in their vertebrate hosts. Numerous outbreaks have been brought on by alphaviruses worldwide in the last few decades. Chikungunya virus (CHIKV) stands out as a major global health concern due to recurrent outbreaks and debilitating chronic arthralgia caused by CHIKV infection. The arthralgia might be due to the persistent replication of CHIKV. CHIKV non-structural protein 4 (nsP4) is the RNA-dependent RNA polymerase (RdRp), which enables the replication of the viral genome and therefore plays a critical role in virus infection. In this work, we established the inducible stable cell lines for CHIKV nsP4 expression. NsP4 expressed in these cells can functionally trans-complement CHIKV mutant genomes harboring lethal mutations in nsP4 or lacking the nsP4 region entirely. This system enables the production of CHIKV virions that are infectious for nsP4 expressing cell lines but limited to early stages of infection (attachment, internalization, and replicase protein expression) in other cells. Moreover, our system was found to be suitable for the analysis of compounds inhibiting CHIKV replication; for example, RdRp inhibitor 4’-Fluorouridine significantly inhibited viral replication in nsP4 expressing cells. Additionally, our study revealed that CHIKV nsP4 can form functional replicases with P123 polyproteins of heterologous alphaviruses using trans-replication assay. This finding expands the versatility of the CHIKV nsP4 stable cell lines. We extended these findings and constructed inducible cell lines that express both nsP1 and nsP4 components of replicase and demonstrated that these cells can complement for lethal defects introduced into both nsP1 and nsP4 of the CHIKV genome. Taken together, CHIKV nsP4 inducible stable cell lines are valuable tools for alphavirus research, can be used to produce virions that are infections only in specific cell lines and therefore be handled without biosafety risk, such tools open new possibilities for studies of alphavirus replication as well as for screening and analysis of inhibitors of alphavirus replication.