Towards understanding the neurovirulence of Semliki Forest virus
Date
2015-11-16
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Abstract
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.
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.
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Keywords
alfaviirus, Semliki metsaviirus, virulentsus, mittestruktuursed valgud, replikatsioon, interaktsioonid, alphavirus, Semliki forest virus, virulence, nonstructural proteins, replication, interactions