Semliki Forest viiruse replikaasivalgu nsP1 uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Semliki Forest viirus kuulub alfaviiruste perekonda sugukonnas Togaviridae. Alfaviirused on olulised patogeenid ning põhjustavad erinevaid vaevusi nii loomadele kui ka inimestele. Levik looduses toimub sääskede vahendusel. Lisaks tähtsusele patogeensuse tõttu on alfaviirused kasutusel ka bio- ning geenitehnoloogias. Alfaviirustel on positiivse polaarsusega RNA genoom, millelt transleeritakse viiruse replikaasi subühikud (nsP1-nsP4). Antud töö põhiline uurimisobjekt nsP1 vastutab lisaks osalemisele replikatsioonil ja transkriptsioonil replikaasi sidumise eest peremeesraku membraanidele. Antud töö tulemusena tuvastati, et nsP1 palmitüleerimine membraaniga seondumise tugevdamiseks ei ole viirusele hädavajalik. Palmitüleerimine hõlbustab kontaktide loomist nsP1 ja viiruse polümeraasi (nsP4) vahel imetaja rakkudes. Palmitüleerimise puudumisel omandab viirus sekundaarsed mutatsioonid, mis taastavad vastavate kontaktide moodustamise võimet imetajarakkudes. Samad mutatsioonid viiruse elulemust putuka rakkudes olulisel määral ei mõjuta. Leiti, et mittestruktuurse polüproteiini lõikamine nsP1 ja nsP2 vahelt on üks funktsionaalse replikaaskompleksi moodustumise kontrollpunktidest. 1/2 saidi lõikamise kiirendamine vähendab olulisel määral viiruse infektsioonilisust; lõikamise aeglustamisel on väiksem mõju. Mutatsioonanalüüs näitas, et 1/2 saidi lõikust mõjutab eelkõige üks nsP1 valgus lõpus asuv aminohappejääk (P5). Täiendav mutatsioon nsP2 kodeerivas piirkonnas (Q706R) taastas mutantse lõikesaidiga viiruse elujõulisuse ja liitvalgu korrektse lõikamise imetaja rakkudes, ent selline viirus ei suutnud edukalt paljuneda putuka rakkudes ning terviklikku interferoonvastust omavates imetaja rakkudes. Nendest tulemustes lähtuvalt pakuti välja hüpotees, mille kohaselt mutatsioonid 1/2 lõikesaidis ja nsP2 valgus põhjustavad viiruse P123 liitvalgu eluea pikenemise, mis takistab viiruse võimet blokeerida natiivse immuunvastuse teket. Uuritud mutatsioonide bioloogiliste efektide põhjalikumaks uurimiseks on tarvis läbi viia loomkatseid.Semliki Forest virus is a member of the family Togaviridae genus Alpavirus. Alphaviruses are pathogens that cause illness in animals and humans. In nature mosquitos transmit the pathogen. In addition to the importance as pathogens alphaviruses are widely used as biotechnological tools. Alphaviruses have a RNA genome with positive polarity and the replicase subunits (nsP1-nsP4) are translated directly from the genome. nsP1, the protein of investigation in the current thesis, is involved in regulating the formation of a replicase complex and attaching the complex to host cell membranes. In the current thesis it was detected that the palmitoylation of nsP1 as a mean for strengthening the interaction with membranes is not indispensable for the virus. In mammalian cells palmitoylation enables direct contacts to be formed between nsP1 and the viral polymerase. If nsP1 is not palmitoylated, the virus acquires second-site mutations that rescue the ability to form contacts between these proteins. The viability of the virus in insect cells is not much affected by same compensatory mutations. The analysis of the processing requirements between nsP1 and nsP2 in the SFV ns-polyprotein highlighted its importance as one of the major regulatory points in the assembly of a functional replication complex. The accelerated processing of this site severely diminished the infectivity of the corresponding mutant genome, while a reduction in the cleavage efficiency had only a minor effect. The processing of the 1/2 site depends mostly on the amino acid residue in the terminal region of nsP1 (P5). An additional mutation (Q706R) in the nsP2 coding region rescued the viability of the virus with the mutant cleavage site and the correct polyprotein processing. The resulting viruses failed to replicate as efficiently as in mammalian cells in both insect cells and mammalian cells with an intact IFN system. It was suggested that the prolonged stabilty of polyprotein P123 that results from the mutation in the P5 position in the 1/2 cleavage site and the Q706R mutation in the nsP2 coding region contributes to the enhanced native immune response in these cells. Detailed studies using animal models are required to provide further information about the biological implications of these findings

Properties and use of novel replication-competent vectors based on Semliki Forest virus

<p>Abstract</p> <p>Background</p> <p>Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important.</p> <p>Results</p> <p>We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the <it>in vitro </it>transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture.</p> <p>Conclusion</p> <p>The properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.</p

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents

Effects of an in-frame deletion of the 6k gene locus from the genome of Ross River virus

The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development

Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1–4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1–nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1–nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots.

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates

Effects of an in-frame deletion of the 6k gene locus from the genome of Ross River virus

The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV 6k deletion mutant. The absence of the 6k gene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.Office of the Snr Dep Vice Chancellor, Institute for GlycomicsFull Tex

Effects of an In-Frame Deletion of the 6k

The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV 6k deletion mutant. The absence of the 6k gene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.Office of the Snr Dep Vice Chancellor, Institute for GlycomicsFull Tex