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Provider: - Institution: - Data provided by Europeana Collections- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain ...Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
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Provider: - Institution: - Data provided by Europeana Collections- Human cytomegalovirus (HCMV) is a ubiquitous human pathogen responsible for devastating
congenital disease and life-threatening ...complications in immune-suppressed patients.
Available treatments have many shortcomings and effective vaccine is still lacking. Major
obstacles to progress in vaccine and antiviral drug development are (1) species specificity of
HCMV, and (2) gaps in our understanding of viral genes and their interaction with host genes.
First limitation is circumvented by the use of model animal viruses, especially murine
cytomegalovirus (MCMV). We sought to alleviate the second problem by studying MCMV
transcriptome using two approaches: classical cDNA library analysis and next generation
sequencing (RNASeq). This dual analysis revealed incredible complexity of MCMV
transcriptome, detected numerous novel viral spliced and unspliced transcripts as well as
transcription from intergenic regions, and showed that expression levels of viral transcripts
vary by several orders of magnitude. Unexpectedly, most top expressed genes were of
unknown functions and were improperly annotated. Therefore, this analysis provides the first
comprehensive overview of MCMV transcriptome, underscores the necessity of
transcriptomic analyses in providing evidence-based genome annotation and could serve as
the first step towards re-annotation of MCMV genome. The most abundant viral transcript,
recently identified as a noncoding RNA regulating cellular microRNAs 18, 84, was shown
to also code for a novel protein(s). This is the first viral transcript that functions both as a
noncoding RNA and an mRNA. In this work it is also shown that this transcript’s 5’ UTR
plays a role in NK cell recognition of infected cells via activating Ly49 receptors.
Analysis of host transcriptome showed that lytic infection revealed that many unexpected
gene groups are disregulated in response to the infection. Such systematic analysis may shed
new light on cytomegalovirus pathogenesis and suggests new areas of research.- Svrha istraživanja
Humani citomegalovirus široko je rasprostranjen patogen, a posebno je opasan za trudnice,
novorođenčad i imunosuprimirane pacijente. Nažalost, efikasnog cjepivo nema, a postoji
potreba i za efikasnijim i manje toksičnim antiviralnim lijekovima. Glavne prepreke razvoju
novih antiviralnih ljekova i cjepiva jesu: (1) specifičnost za vrstu i (2) praznine u našem
znanju i razumjevanju virusnih gena, interakcijama virusnih gena i domaćina te kako te
interakcije izazivaju bolest. Prva prepreka uspješno se nadvladava korištenjem animalnih
virusa, posebice mišjeg citomegalovirusa (MCMV). S ciljem nadvladavanja druge prepreke u
ovom je radu provedena detaljna analiza transkriptoma mišjeg citomegalovirusa (MCMV) te
analiza transkriptoma stanica domaćina tijekom litičke infekcije citomegalovirusom.
Materijali i metode
Transkriptom MCMV analiziran je na dva načina: klasičnom analizom cDNK knjižnice i
sekvencioniranjem dobivenih klonova te analizom transkriptoma uz pomoć sekvencioniranja
nove generacije (odnosno RNK-sekvencioniranjem, eng. RNASeq) koja omogućava paralelno
praćenje i transkriptoma domaćina uz transkriptom virusa. Analiza transkriptoma domaćina
rezultira vrlo dugačkim listama diferencijalno reguliranih gena iz kojih je teško izvući neko
biološko značenje. Stoga su liste diferencijalno reguliranih gena domaćina podvrgnute analizi
termina genske ontologije (eng. gene ontology analysis odnosno GO analiza) i analizom dereguliranih
bioloških puteva. Transkripcijski kompleksne regije genoma MCMV dodatno su
analizirane Northern hibridizacijom i metodom RT-PCR dok je korelacija između količine
transkripata i proteina odabranih gena analizirana metodom Western blot. Na kraju, funkcija
novog, prekrojenog transkripta MAT (most abundant transcript; najzastupljeniji transkript)
analizirana je uz pomoć reporterskih stanica koje nose aktivacijske Ly49 receptore.
Rezultati
Ovaj rad predstavlja prvu detaljnu analizu transkriptoma MCMV-a korištenjem
komplementarnih metoda analize cDNK knjižnice i RNASeq analizom, a rezultirala je
identifikacijom brojnih novih transkripata MCMV, uključujući i nove prekrojene transkripte
kao i transkripte koji se prepisuju sa intergenskih regija. Ustanovljeno je da najjače izraženi
virusni transkripti imaju nepoznatu funkciju i često su pogrešno anotirani. Najjače izražen
transkript (tzv. MAT transkript) prvi je virusni transkript koji ima i kodirajuću i nekodirajuću
funkciju. Naime, nedavno je pokazano da se na njegovom 3' netranslatiranom kraju (3' UTR,
od eng. 3' untranslated region) nalazi vezno mjesto za staničnu mikro-RNK 18, 84, dok je u
ovom radu pokazano da on također kodira i barem još dva proteina. Uz ove dvije navedene
funkcije, u ovom je radu otkriveno da je 5' netranslatirani kraj (5'UTR) MAT transkripta bitan
virusni faktor kojeg na inificranim stanicama prepoznaju stanice prirodne ubojice pomoću
aktivacijskih receptora Ly49.
Analiza transkriptoma stanica domaćina pokazala je da litička infekcija virusom MCMV
izaziva izrazite promjene u ekspresijskom profilu gena domaćina: ekspresija gotovo trećine
gena miša promijenila se uslijed infekcije virusom MCMV. Geni čija se ekspresija pojačava
tijekom infekcije uglavnom su geni uključeni u upalne i imunološke procese, međutim neki
pripadaju i skupini transkripcijskih faktora te genima povezanim s razvojem i
diferencijacijom. Ovi rezultati u skladu su s dosadašnjim saznanjima o CMV-u kao virusu
koji izaziva upalu te uzrokuje razvojne poremećaje tijekom kongenitalnih infekcija. Brojni
geni čija se ekspresija smanjila tijekom infekcije povezani su sa funkcijama čija je uloga u
infekciji za sada nepoznata poput dugačkih intergenskih nekodirajućih RNK, antisense RNK
ili malih nukleolarnih RNK. GO analiza rezultirala je detekcijom disreguliranih bioloških
puteva koji još do sada nisu bili povezani sa citomegalovirusnom infekcijom, a koji imaju
potencijal rasvjetljavanja nekih nepoznanica u patogenezi citomegalovirusne infekcije.
Zaključci
Jedno od najznačajnijih otkrića u ovome radu jest dokaz izuzetne kompleksnosti
transkriptoma MCMV-a koji dosad nije bio ovako sustavno istraživan niti su postojale
transkriptomske mape. Ova analiza transkriptoma MCMV-a predstavlja važan prvi korak ka
razvoju boljih genomskih mapa i reanotaciji genoma MCMV-a. Analiza odgovora stanica
domaćina na infekciju dala je novi pogled na molekularne interakcije između virusa i
domaćina i otvorila brojna nova područja istraživanja koja imaju potencijal da p pronađu nove
mogućnosti liječenja bolesti izazvanih CMV-om.
Ključne riječi
mišji citomegalovirus, MCMV, transkriptom, ekspresija gena, izbjegavanje imunološkog- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
