Samm lähemale mõistmaks Semliki Forest viiruse neurovirulentsust

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Semliki Forest viirus (SFV) on positiivse polaarsusega RNA genoomiga viirus, mis kuulub alfaviiruste perekonda sugukonnas Togaviridae. Alfaviiruste seas leidub mitmeid inimese ja loomade patogeene, mille poolt põhjustatavad haigused varieeruvad külmetuse sarnaste sümptomitega tõvest aastaid kestva artriidi või fataalse entsefaliidini. SFV-d on laialdaselt kasutatud nii alusuuringutes kui ka mudelobjektina viirusliku entsefaliidiga kaasneva patogeneesi uurimisel. Kõige põhjalikumalt uuritud SFV tüvedeks on A7(74), SFV4 ja L10. A7(74) on avirulentne tüvi, mis põhjustab täiskasvanud hiires asümptomaatilist infektsiooni. SFV4 ja L10 on aga virulentsed tüved, sest otse ajju või kõrge doosiga kõhuõõnde süstituna põhjustavad nad hiirtel surmaga lõppevat entsefaliiti. Madalama viiruse koguse kasutamise korral ei jõua SFV4 ajju ning nakatatud katseloomad jäävad ellu. Käesolev uurimistöö näitas, et SFV4 ja L10 vahelised erinevused hiires on tingitud viiruse ümbrisevalgu (E2) aminohappejääkide laengutest. Positiivse laenguga lüsiinijäägid E2 valgus soodustavad küll viiruse seondumist koekultuuri rakkudele, kuid põhjustavad in vivo tingimustes SFV4 virionide efektiivse seostumise raku pinnaretseptori heparaansulfaadiga, mis vähendab viiruse taset veres ja selle kaudu võimekust ajju siseneda. Me näitasime, et erinevused A7(74) ja L10 neurovirulentsuses on tingitud erinevustest viiruse mittestruktuurse liitvalgu proteolüütilise lõikamise kiiruses ning mittestruktuurse valgu 3 (nsP3) järjestuses. P123 liitvalgu 1/2 lõikamisjärjestuse aeglane protsessimine või L10 nsP3 valgu olemasolu suurendavad SFV replikaasi võimet indutseerida interferooni tootmist, korreleerudes viiruse võimega levida hiire ajus, mis viitab seosele SFV neurovirulentsuse ja immunopatoloogia vahel. Viiruse ja peremehe vahel esinevate keeruliste interaktsioonide veel täpsemaks uurimiseks arendasime välja uue lähenemise, mis võimaldas tuvastada SFV replikatsioonikompleksidega seonduvaid rakulisi valke. Käesoleva uurimistöö käigus saadud tulemused aitavad ühelt poolt paremini mõista SFV neurovirulentsust, lisaks sellele on mudelviiruse uurimisel kasutatud meetode ja avastatud seaduspärasusi võimalik rakendada ka inimesele ohtlike alfaviiruste uurimiseks.Semliki Forest virus (SFV) is positive-sense RNA virus belonging to the genus Alphavirus in the family Togaviridae. This genus includes viruses pathogenic to a wide variety of animals, including humans, causing a spectrum of diseases that ranges from unpleasant flu-like illness and arthritis to fatal encephalitis. Laboratory strains of SFV have been utilized extensively in genetic engineering and they provide a well-characterized model system to investigate the pathogenesis of viral encephalitis. The most thoroughly studied strains of SFV are A7(74), SFV4, and L10. A7(74) is considered to be avirulent because the infection in adult mice is asymptomatic. In contrast, after intracerebral or high-dose intraperitoneal (i.p.) inoculation, L10 and SFV4 are both virulent and cause lethal encephalitis. However, following low-dose i.p. inoculation, SFV4 is incapable of reaching the brain, and the infected animals survive. Current thesis showed that phenotypic differences between SFV4 and L10 are determined by the charge of amino acid residues in viral glycoprotein E2. Positively charged amino acid residues in E2 facilitate the binding of SFV4 virions to heparan sulfate on the cell surface. This results in rapid clearance of virus from the blood and lower viremia, which in turn prevents the entry of the virus into the brain. We showed that phenotypic differences between A7(74) and L10 are caused by differences in the rate of nonstructural polyprotein processing and in the sequence of nonstructural protein 3 (nsP3). Slower processing of the polyprotein P123 1/2 cleavage site or the presence of L10 nsP3 enhance the ability of the virus replicase to induce interferon production, which correlates with virus ability to spread in the brain, suggesting a link between SFV neurovirulence and immunopathology. To further analyze the intricate interplay between the virus and the host, we developed a novel approach to determine the host proteins that colocalize with mature replication complexes of SFV. These data together help us better understand the neurovirulence of SFV. Furthermore, the methods and mechanisms discovered here can be applied in the research of medically important alphaviruses

Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC50] = 0.03 mu M) and Semliki Forest virus (SFV; EC50 = 0.11 mu M). Obatoclax inhibited viral entry processes in an SFV temperaturesensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate.Peer reviewe

Mutation of CD2AP and SH3KBP1 binding motif in alphavirus nsP3 hypervariable domain results in attenuated virus

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3

Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.Peer reviewe

Obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses including chikungunya virus, West Nile virus, influenza virus etc. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins was previously determined to be antiviral against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses like chikungunya virus (EC50 = 0.03 μM) and Semliki Forest virus (SFV) (EC50 = 0.11 μM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. Neutral red retention assay revealed that obatoclax induces rapid neutralization of the acidic environment of endolysosomal vesicles and thereby, most likely inhibits viral fusion. Characterization of escape mutants revealed that mutation L369I in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins neither inhibited viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses like Zika virus, West Nile virus and yellow fever virus, which require low pH for fusion, but not of pH-independent picornaviruses like coxasackievirus A9, echovirus 6 and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification preventing viral fusion that could be pursued as a potential broad-spectrum antiviral candidate.</p

Magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of Semliki Forest virus

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection

Expression of marker proteins in cell lines transfected with <i>trans</i>-replicase vectors.

<p>Huh7, U2OS, COP-5, BHK-21 and BSR cells were co-transfected with CMV-P1234 + CMV-Fluc-Gluc (marked as CMV); BSR cells were also co-transfected with T7-P1234 + T7-FLuc-Gluc (marked as T7). Control cells were transfected with T7-FLuc-Gluc or CMV-Fluc-Gluc together with corresponding plasmid encoding for inactive replicase. Cells were lysed at 18 h post transfection. Gluc <b>(A)</b> and Fluc <b>(C)</b> activities (RLU—relative light unit), normalized to the total protein content in the lysate, are shown. Each column represents an average of three independent experiments; error bars represent standard deviation. All differences between P1234^GAA (empty columns) and P1234 (grey columns) were highly significant (p<<0.001); in C ** designates p<0.01 and *** designates p<0.001 (one-way ANOVA Sidak’s multiple comparisons test). <b>(B)</b> and <b>(D)</b>. The reporter activities generated by the active replicase were normalized to those measured with the inactive controls.</p

Tagging of replicase expression constructs.

<p><b>(A)</b> Positions used for EGFP insertion are indicted by arrows with numbers corresponding to the amino acid residue after which the EGFP was inserted. Tags fused to the C-terminus of nsP4 are designated as follows: E—EGFP; HF—HA-tag+3xFLAG-tag; SF—Strep-tag+3xFLAG-tag; HS—two copies of HA-tag+Strep-tag. <b>(B)</b> BSR cells were co-transfected with T7-Fluc-Gluc and T7-P1234 or plasmids expressing tagged versions of ns polyprotein (indicated below the graphs). Cells co-transfected with T7-Fluc-Gluc + T7-P1234^GAA served as controls. At 18 h post transfection cells were lysed, and activities of Fluc (left panel) and Gluc (right panel) in the cell lysates were measured. The activities of reporters were normalized to those measured in control cells. Data from one independent reproducible experiment out of three is shown. <b>(C)</b> U2OS cells were co-transfected with CMV-Fluc-Gluc and CMV-P1234 or plasmids expressing tagged versions of ns polyprotein. The data is presented as in B. <b>(D)</b> BSR (left) and U2OS (right) cells were used to detect expression of ns proteins from T7- and CMV promoter-based vectors, respectively. The constructs used are indicated at the top. Analysis was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151616#pone.0151616.g003" target="_blank">Fig 3A</a>; additionally anti-EGFP and anti-FLAG antibodies were also used for detection of proteins carrying corresponding tags. Data from one independent reproducible experiment out of two is shown.</p