Title MOLEKULARNA I FUNKCIONALNA KARAKTERIZACIJA
REGIJE M116 MIŠJEG CITOMEGALOVIRUSA Title (english) MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF
M116 REGION IN MOUSE CYTOMEGALOVIRUS Author Tina Ružić Mentor Berislav Lisnić (mentor)Committee member Ilija Brizić (predsjednik povjerenstva)Committee member Helena Ćetković (član povjerenstva)Committee member Đurđica Cekinović Grbeša (član povjerenstva)Granter University of Rijeka Defense date and country 2022, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences ThesaurusMESH - Medical Subject Headings )
Cytomegalovirus
Cytomegalovirus Infections
Muromegalovirus
pathogenicity
Herpesviridae Infections
Mononuclear Phagocyte System
metabolism
Abstract Cilj istraživanja: Humani citomegalovirus (HCMV) herpesvirus je specifičan za vrstu koji
uzrokuje tešku bolest u osoba s oslabljenim ili nezrelim imunosnim sustavom. Mišji
citomegalovirus (MCMV) biološki je i genetički vrlo srodan HCMV-u te služi kao općeprihvaćen
model za istraživanje infekcije, patogeneze i imunosnog odgovora na citomegaloviruse.
Prethodna istraživanja naše grupe pokazala su da su najviše ispoljene regije genoma MCMVa slabo karakterizirane. Jedna od njih je regija M116 koja kodira za produkt nepoznate
strukture i funkcije, a homologna je regiji UL116 HCMV-a. Stoga je cilj ovog istraživanja
provesti molekularnu i funkcionalnu karakterizaciju regije M116 MCMV-a.
Materijali i metode: Kako bi karakterizirali transkripte regije M116 koristili smo metode
Northern blot te RACE analizu. Za detekciju potencijalnih proteina kodiranih regijom M116
razvili smo i testirali monoklonsko protutijelo α-M116 koje smo uspješno primijenili za detekciju
i karakterizaciju proteina M116.1 u svim korištenim tehnikama: Western blot analizi, protočnoj
citometriji, imunohistokemiji, konfokalnoj mikroskopiji, testu neposredne ligacije i
imunoprecipitaciji. Kako bi mogli istraživati regiju M116 i razumjeti ulogu produkata ove regije
u patogenezi i životnom ciklusu MCMV-a, konstruirali smo virus kojemu nedostaje najviše
ispoljen dio regije M116 (ΔM116-MCMV). Ispitali smo razlike u in vitro infekciji fibroblasta i
mononuklearnih fagocita između rekombinantnog virusa ΔM116-MCMV i virusa divljeg tipa
WT-MCMV te karakterizirali virus ΔM116-MCMV in vivo infekcijom BALB/c miševa.
Rezultati: Genski lokus M116 MCMV-a kodira za dva kasno ispoljena, 5'-koterminalna,
prekrojena i poliadenilirana transkripta M116-M114 i M116 koja kodiraju protein M116.1.
Protein M116.1 očuvan je među različitim sojevima MCMV-a, N-glikoziliran i ispoljen kasnom
kinetikom, a lokalizira se u vanjskom prstenu citoplazmatskog odjeljka za sklapanje viriona
gdje stupa u interakciju s proteinom gH, potencijalno vršeći ulogu njegovog šaperona analogno
homolognom proteinu UL116 HCMV-a. Protein M116.1 neophodan je za efikasnu infekciju
mononuklearnih fagocita in vitro. Nedostatak proteina M116.1 nema značajnog utjecaja na
širenje virusa uslijed intravenozne infekcije ili infekcije u šapicu, ali umanjuje sposobnost
MCMV-a da se uspješno širi nakon intranazalne infekcije.
Zaključak: U ovom je istraživanju po prvi puta karakteriziran lokus M116, njegovi transkripti i
protein M116.1. Rezultati ukazuju na važnost proteina M116.1 za uspješnost infekcije
mononuklearnih fagocita MCMV-om te njegovog utjecaja na tropizam i širenje virusa u
kontekstu različitih ruta infekcije, a ujedno i doprinose razumijevanju funkcije proteina UL116
u HCMV-u.
Abstract (english) Objectives: Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes
severe disease in immunocompromised individuals and immunologically immature neonates.
Murine cytomegalovirus (MCMV) is biologically similar to HCMV and serves as a widely used
model for studying the infection, pathogenesis, and immune responses to HCMV. In our
previous work, we have identified the M116 gene region as one of the most extensively
transcribed regions of the MCMV genome without an assigned function.
Material and methods: To characterize the transcripts of the M116 region, we performed
Northern blot and RACE analysis. We developed and tested new α-M116 monoclonal
antibody, which was then used for detection and characterization of M116.1 protein in multiple
techniques: Western blot analysis, flow cytometry, immunohistochemistry, confocal
microscopy, proximity ligation assay and immunoprecipitation. To investigate the M116 region
and understand its role in the pathogenesis and life cycle of MCMV, we constructed a MCMV
that lacks the most abundantly expressed portion of the M116 region (ΔM116-MCMV). We
examined differences in in vitro infection of fibroblasts and mononuclear phagocytes between
recombinant ΔM116-MCMV and WT-MCMV and characterized ΔM116-MCMV during in vivo
infection of BALB/c mice.
Results: The MCMV M116 gene locus encodes two late, 5'-coterminal, spliced, and
polyadenylated transcripts: M116-M114 and M116, both encoding the M116.1 protein. Protein
M116.1 is conserved among different strains of MCMV, N-glycosylated, expressed with late
kinetics and localized to the outer ring of the cytoplasmic virion assembly compartment where
it interacts with gH, potentially acting as its chaperone in a similar fashion to homologous
HCMV protein UL116. Although protein M116.1 was necessary for efficient infection of
mononuclear phagocytes in vitro, its deficiency had no significant effect on virus spread during
intravenous or footpad infection. However, we observed reduced ability of ΔM116-MCMV to
spread successfully upon intranasal infection.
Conclusion: This study was the first to characterize the M116 locus, its transcripts, and the
M116.1 protein. The results indicate the importance of M116.1 protein for the efficient infection
of mononuclear phagocytes with MCMV and its impact on tropism and virus spread in the
context of different routes of infection, but also contributes to the understanding of UL116
protein function in HCMV.
Keywords
Humani Citomegalovirus
M116
Mišji Citomegalovirus
Mononuklearni fagociti
Tropizam
UL116
Keywords (english)
Human Cytomegalovirus
M116
Mononuclear Phagocyte System
Mouse Cytomegalovirus
Tropism
UL116
Language croatian URN:NBN urn:nbn:hr:184:896490 Promotion 2022 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2022-07-04 10:18:30
