Cytoplasmic virion assembly compartment of betaherpesviruses

During their life cycle, betaherpesviruses extensively reorganize the membranous system of the cell in order to develop a unique cytoplasmic environment of virion production ("virus factory"), known as cytoplasmic virion assembly compartment (cVAC). The betaherpesvirus cVAC has been studied both in human and murine cytomegalovirus (CMV) infected cells as they are considered to be models for betaherpesviruses biology. Studies of the cVAC were mainly based on electron microscopy and immunofluorescence tools, and in the previous decade, they have expanded to the use of new technologies and systems approaches (i.e., transcriptomics, proteomics, lipidomics), which led to the acquisition of a vast amount of data. Despite the immense expansion of knowledge about membranous organelle system, including a large amount of data derived from CMV infected cells, the organization and biogenesis of the cVAC remain unclear. Accordingly, very little is known about the processes of a final stage of CMV maturation and the virion egress. This minireview summarizes current knowledge about the biogenesis of the betaherpesvirus factory from the already existent endosomal compartments. &nbsp;</p

EFFECT OF EARLY PHASE MOUSE CYTOMEGALOVIRUS INFECTION ON REMODELLING ENDOSOMAL SYSTEM IN FIBROBLASTS

Cilj istraživanja: U ovom radu cilj je bio razotkriti mehanizam kojim virus preuređuje sustav endosoma tijekom rane faze infekcije MCMVom. Stoga smo istražili funkciju proteaza te glavnih endosomalnih putova u inficiranim stanicama, kao i njezin utjecaj na unutarstanični izražaj Rab i Arf obitelji regulatornih bjelančevina. Materijal i metode: U istraživanjima smo koristili dvije mišje fibroblastne linije: primarne mišje embrionalne fibroblaste (MEF) i imortalizirane Balb3T3 stanice te dva rekombinantna soja virusa: Δm138-MCMV i Δ9-MCMV. Smještaj ispitivanih staničnih i virusnih bjelančevina pratili smo imunofluorescencijom i konfokalnom mikroskopijom. Stanične kinetičke parametre smo određivali prvenstveno protočnom citometrijom (uz korištenjem softwarea izrađenog u suradnji sa Tehničkim fakultetom Sveučilišta u Rijeci). Promjene u razini izražaja pojedinih biljega različitih staničnih segmenata određivali smo Western blot analizom. Molekule su praćene bez ili nakon primijene različitih kemijskih inhibitora. Rezultati: Za razvoj infekcije MCMVom potreban je funkcionalan sustav staničnih serinskih proteaza koje djeluju u neutralnom pH, koje uključuju furin. Funkcionalnim testovima smo pokazali da u ranoj fazi infekcije dolazi do promjena u brzinama prijenosa transferina, molekula MHC-I i sfingomijelina kroz stanični sustav endosoma. Njihovo recikliranje je usporeno kao posljedica infekcije, a molekule su zadržane perinuklearno, u virusnom remodeliranom EPERC odjeljku. Matematičkim modeliranjem smo pokazali da je u inficiranim stanicama održano brzo recikliranje, a da je inhibiran izlazak i iz sortirajućih endosoma (SE) i iz endosomalnog reciklirajućeg odjeljka (ERC), kao i prijenos iz SE u ERC. Razlog zastoja u izlasku iz SE, kao i iz ERCa je najvjerojatnije posljedica utjecaja MCMVa na razinu izražaja i aktivnost staničnih regulacijskih bjelančevina Rab i Arf obitelji (Arf6, Rab35 i Rab22A), što smo pokazali konfokalnom mikroskopijom i Western blot analizom. Zaključak: Za normalan razvoj infekcije potrebne su serinske proteaze (npr. furin). Infekcijom MCMVom dolazi do kočenja izlaska endocitiranog materijala iz SE i ERCa, što je najvjerojatnije prouzročeno utjecajem MCMVa na regulacijske bjelančevine Rab i Arf obitelji (Arf6, Rab35 i Rab22A.Aim of the study: This study aimed to elucidate mechanisms that rearrange endosomal system in the early phase of MCMV infection. Therefore, we investigated the function of cellular proteases and major endosomal routes in infected cells as well the MCMV influence in the intracellular expression levels of regulatory protein families Rab and Arf. Materials and methods: For our experiments, we used murine embryonic fibroblasts (MEF) and immortalized fibroblast-like Balb3T3 cells and two recombinant MCMV viruses: Δm138-MCMV and Δ9-MCMV. Intracellular distribution and expression of endosomal regulatory proteins, endosomal marker proteins, and viral proteins, was analyzed by immunofluorescence techniques and imaging by confocal microscopy. Cellular kinetic parameters were determined by flow cytometry (using in-house developed software, made in collaboration with Faculty of Engineering of the University of Rijeka). The intracellular expression levels of endosomal markers and viral proteins were quantified by Western blot. Molecules were followed with or without the application of different chemical inhibitors. Results: Functional system of cellular proteases that are active in the neutral pH environment (including furin), is required for the development of MCMV infection. Functional assays revealed alteration of the endosomal flow in the early phase of MCMV infection, as demonstrated by several cargo molecules, including trafficking of TfR, MHC-I molecules and NBD-sphingomyelin through the cellular endosomal system. Their recycling was delayed, and cargo molecules were retained in EPERC, the perinuclear endosomal compartment remodeled by MCMV. The kinetic modeling revealed that MCMV infection does not affect the rapid recycling route, but inhibits the egress from sorting endosomes (SE) and endosomal recycling compartment (ERC) as well as transfer form SE to ERC. The inhibition of the egress from SEs and the ERC was associated with altered levels and subcellular distribution of cellular regulatory proteins, most importantly Arf6, Rab35, and Rab22A (demonstrated by the immunofluorescence imaging and Western blot analysis). Conclusion: Serine proteases (i.e. furin) are necessary for the regular development of infection. MCMV infection causes retention of endocytosed cargo in SEs and the ERC which is associated by redistribution of the small GTPases from Rab and Arf families (Arf6, Rab35, and Rab22A

EFFECT OF EARLY PHASE MOUSE CYTOMEGALOVIRUS INFECTION ON REMODELLING ENDOSOMAL SYSTEM IN FIBROBLASTS

Cilj istraživanja: U ovom radu cilj je bio razotkriti mehanizam kojim virus preuređuje sustav endosoma tijekom rane faze infekcije MCMVom. Stoga smo istražili funkciju proteaza te glavnih endosomalnih putova u inficiranim stanicama, kao i njezin utjecaj na unutarstanični izražaj Rab i Arf obitelji regulatornih bjelančevina. Materijal i metode: U istraživanjima smo koristili dvije mišje fibroblastne linije: primarne mišje embrionalne fibroblaste (MEF) i imortalizirane Balb3T3 stanice te dva rekombinantna soja virusa: Δm138-MCMV i Δ9-MCMV. Smještaj ispitivanih staničnih i virusnih bjelančevina pratili smo imunofluorescencijom i konfokalnom mikroskopijom. Stanične kinetičke parametre smo određivali prvenstveno protočnom citometrijom (uz korištenjem softwarea izrađenog u suradnji sa Tehničkim fakultetom Sveučilišta u Rijeci). Promjene u razini izražaja pojedinih biljega različitih staničnih segmenata određivali smo Western blot analizom. Molekule su praćene bez ili nakon primijene različitih kemijskih inhibitora. Rezultati: Za razvoj infekcije MCMVom potreban je funkcionalan sustav staničnih serinskih proteaza koje djeluju u neutralnom pH, koje uključuju furin. Funkcionalnim testovima smo pokazali da u ranoj fazi infekcije dolazi do promjena u brzinama prijenosa transferina, molekula MHC-I i sfingomijelina kroz stanični sustav endosoma. Njihovo recikliranje je usporeno kao posljedica infekcije, a molekule su zadržane perinuklearno, u virusnom remodeliranom EPERC odjeljku. Matematičkim modeliranjem smo pokazali da je u inficiranim stanicama održano brzo recikliranje, a da je inhibiran izlazak i iz sortirajućih endosoma (SE) i iz endosomalnog reciklirajućeg odjeljka (ERC), kao i prijenos iz SE u ERC. Razlog zastoja u izlasku iz SE, kao i iz ERCa je najvjerojatnije posljedica utjecaja MCMVa na razinu izražaja i aktivnost staničnih regulacijskih bjelančevina Rab i Arf obitelji (Arf6, Rab35 i Rab22A), što smo pokazali konfokalnom mikroskopijom i Western blot analizom. Zaključak: Za normalan razvoj infekcije potrebne su serinske proteaze (npr. furin). Infekcijom MCMVom dolazi do kočenja izlaska endocitiranog materijala iz SE i ERCa, što je najvjerojatnije prouzročeno utjecajem MCMVa na regulacijske bjelančevine Rab i Arf obitelji (Arf6, Rab35 i Rab22A.Aim of the study: This study aimed to elucidate mechanisms that rearrange endosomal system in the early phase of MCMV infection. Therefore, we investigated the function of cellular proteases and major endosomal routes in infected cells as well the MCMV influence in the intracellular expression levels of regulatory protein families Rab and Arf. Materials and methods: For our experiments, we used murine embryonic fibroblasts (MEF) and immortalized fibroblast-like Balb3T3 cells and two recombinant MCMV viruses: Δm138-MCMV and Δ9-MCMV. Intracellular distribution and expression of endosomal regulatory proteins, endosomal marker proteins, and viral proteins, was analyzed by immunofluorescence techniques and imaging by confocal microscopy. Cellular kinetic parameters were determined by flow cytometry (using in-house developed software, made in collaboration with Faculty of Engineering of the University of Rijeka). The intracellular expression levels of endosomal markers and viral proteins were quantified by Western blot. Molecules were followed with or without the application of different chemical inhibitors. Results: Functional system of cellular proteases that are active in the neutral pH environment (including furin), is required for the development of MCMV infection. Functional assays revealed alteration of the endosomal flow in the early phase of MCMV infection, as demonstrated by several cargo molecules, including trafficking of TfR, MHC-I molecules and NBD-sphingomyelin through the cellular endosomal system. Their recycling was delayed, and cargo molecules were retained in EPERC, the perinuclear endosomal compartment remodeled by MCMV. The kinetic modeling revealed that MCMV infection does not affect the rapid recycling route, but inhibits the egress from sorting endosomes (SE) and endosomal recycling compartment (ERC) as well as transfer form SE to ERC. The inhibition of the egress from SEs and the ERC was associated with altered levels and subcellular distribution of cellular regulatory proteins, most importantly Arf6, Rab35, and Rab22A (demonstrated by the immunofluorescence imaging and Western blot analysis). Conclusion: Serine proteases (i.e. furin) are necessary for the regular development of infection. MCMV infection causes retention of endocytosed cargo in SEs and the ERC which is associated by redistribution of the small GTPases from Rab and Arf families (Arf6, Rab35, and Rab22A

Application of Abf2 and MC1 proteins in preparation of short circular DNA for cell transfection

Jednu od metoda molekulske terapije za modificiranje aktivnosti transkripcijskih faktora čine DNA-mamci (duljine manje od 100 pb). Do sada opisani oblici DNA-mamaca lako se degradiraju endonukleazama. Kružne molekule DNA su rezistentne na taj tip degradacije. Ipak, kratke molekule DNA (kraće od oko 150 pb) ne mogu se cirkularizirati samo uz upotrebu DNA-ligaze U ovom radu opisana je prva uspješna cirkularizacija dvolančane molekule DNA duljine 95 pb uz upotrebu proteina za savijanje DNA (Abf2 i MC1). Isto tako je konstruirana kružna DNA upotrijebljena za transfekciju stanica u kulturi.One of the molecular therapy methods for transcription factor activity modification are DNA-decoys (molecules shorter than 100 bp). All types of DNA-decoys that have been described so far are easily degraded by exonucleases. Circular DNA molecules are, however, resistant to exonuclease activity. Also, it is impossible to create circular DNA from linear dsDNA that is relatively shorter than 150 bp. This is the first published work with described circularization of 95 bp long dsDNA by using Abf2 protein and MC1 protein. The constructed circular DNA was used to transfect cells in cell culture

Application of Abf2 and MC1 proteins in preparation of short circular DNA for cell transfection

Jednu od metoda molekulske terapije za modificiranje aktivnosti transkripcijskih faktora čine DNA-mamci (duljine manje od 100 pb). Do sada opisani oblici DNA-mamaca lako se degradiraju endonukleazama. Kružne molekule DNA su rezistentne na taj tip degradacije. Ipak, kratke molekule DNA (kraće od oko 150 pb) ne mogu se cirkularizirati samo uz upotrebu DNA-ligaze U ovom radu opisana je prva uspješna cirkularizacija dvolančane molekule DNA duljine 95 pb uz upotrebu proteina za savijanje DNA (Abf2 i MC1). Isto tako je konstruirana kružna DNA upotrijebljena za transfekciju stanica u kulturi.One of the molecular therapy methods for transcription factor activity modification are DNA-decoys (molecules shorter than 100 bp). All types of DNA-decoys that have been described so far are easily degraded by exonucleases. Circular DNA molecules are, however, resistant to exonuclease activity. Also, it is impossible to create circular DNA from linear dsDNA that is relatively shorter than 150 bp. This is the first published work with described circularization of 95 bp long dsDNA by using Abf2 protein and MC1 protein. The constructed circular DNA was used to transfect cells in cell culture

EFFECT OF EARLY PHASE MOUSE CYTOMEGALOVIRUS INFECTION ON REMODELLING ENDOSOMAL SYSTEM IN FIBROBLASTS

Cilj istraživanja: U ovom radu cilj je bio razotkriti mehanizam kojim virus preuređuje sustav endosoma tijekom rane faze infekcije MCMVom. Stoga smo istražili funkciju proteaza te glavnih endosomalnih putova u inficiranim stanicama, kao i njezin utjecaj na unutarstanični izražaj Rab i Arf obitelji regulatornih bjelančevina. Materijal i metode: U istraživanjima smo koristili dvije mišje fibroblastne linije: primarne mišje embrionalne fibroblaste (MEF) i imortalizirane Balb3T3 stanice te dva rekombinantna soja virusa: Δm138-MCMV i Δ9-MCMV. Smještaj ispitivanih staničnih i virusnih bjelančevina pratili smo imunofluorescencijom i konfokalnom mikroskopijom. Stanične kinetičke parametre smo određivali prvenstveno protočnom citometrijom (uz korištenjem softwarea izrađenog u suradnji sa Tehničkim fakultetom Sveučilišta u Rijeci). Promjene u razini izražaja pojedinih biljega različitih staničnih segmenata određivali smo Western blot analizom. Molekule su praćene bez ili nakon primijene različitih kemijskih inhibitora. Rezultati: Za razvoj infekcije MCMVom potreban je funkcionalan sustav staničnih serinskih proteaza koje djeluju u neutralnom pH, koje uključuju furin. Funkcionalnim testovima smo pokazali da u ranoj fazi infekcije dolazi do promjena u brzinama prijenosa transferina, molekula MHC-I i sfingomijelina kroz stanični sustav endosoma. Njihovo recikliranje je usporeno kao posljedica infekcije, a molekule su zadržane perinuklearno, u virusnom remodeliranom EPERC odjeljku. Matematičkim modeliranjem smo pokazali da je u inficiranim stanicama održano brzo recikliranje, a da je inhibiran izlazak i iz sortirajućih endosoma (SE) i iz endosomalnog reciklirajućeg odjeljka (ERC), kao i prijenos iz SE u ERC. Razlog zastoja u izlasku iz SE, kao i iz ERCa je najvjerojatnije posljedica utjecaja MCMVa na razinu izražaja i aktivnost staničnih regulacijskih bjelančevina Rab i Arf obitelji (Arf6, Rab35 i Rab22A), što smo pokazali konfokalnom mikroskopijom i Western blot analizom. Zaključak: Za normalan razvoj infekcije potrebne su serinske proteaze (npr. furin). Infekcijom MCMVom dolazi do kočenja izlaska endocitiranog materijala iz SE i ERCa, što je najvjerojatnije prouzročeno utjecajem MCMVa na regulacijske bjelančevine Rab i Arf obitelji (Arf6, Rab35 i Rab22A.Aim of the study: This study aimed to elucidate mechanisms that rearrange endosomal system in the early phase of MCMV infection. Therefore, we investigated the function of cellular proteases and major endosomal routes in infected cells as well the MCMV influence in the intracellular expression levels of regulatory protein families Rab and Arf. Materials and methods: For our experiments, we used murine embryonic fibroblasts (MEF) and immortalized fibroblast-like Balb3T3 cells and two recombinant MCMV viruses: Δm138-MCMV and Δ9-MCMV. Intracellular distribution and expression of endosomal regulatory proteins, endosomal marker proteins, and viral proteins, was analyzed by immunofluorescence techniques and imaging by confocal microscopy. Cellular kinetic parameters were determined by flow cytometry (using in-house developed software, made in collaboration with Faculty of Engineering of the University of Rijeka). The intracellular expression levels of endosomal markers and viral proteins were quantified by Western blot. Molecules were followed with or without the application of different chemical inhibitors. Results: Functional system of cellular proteases that are active in the neutral pH environment (including furin), is required for the development of MCMV infection. Functional assays revealed alteration of the endosomal flow in the early phase of MCMV infection, as demonstrated by several cargo molecules, including trafficking of TfR, MHC-I molecules and NBD-sphingomyelin through the cellular endosomal system. Their recycling was delayed, and cargo molecules were retained in EPERC, the perinuclear endosomal compartment remodeled by MCMV. The kinetic modeling revealed that MCMV infection does not affect the rapid recycling route, but inhibits the egress from sorting endosomes (SE) and endosomal recycling compartment (ERC) as well as transfer form SE to ERC. The inhibition of the egress from SEs and the ERC was associated with altered levels and subcellular distribution of cellular regulatory proteins, most importantly Arf6, Rab35, and Rab22A (demonstrated by the immunofluorescence imaging and Western blot analysis). Conclusion: Serine proteases (i.e. furin) are necessary for the regular development of infection. MCMV infection causes retention of endocytosed cargo in SEs and the ERC which is associated by redistribution of the small GTPases from Rab and Arf families (Arf6, Rab35, and Rab22A

Cytoplasmic virion assembly compartment of betaherpesviruses

During their life cycle, betaherpesviruses extensively reorganize the membranous system of the cell in order to develop a unique cytoplasmic environment of virion production ("virus factory"), known as cytoplasmic virion assembly compartment (cVAC). The betaherpesvirus cVAC has been studied both in human and murine cytomegalovirus (CMV) infected cells as they are considered to be models for betaherpesviruses biology. Studies of the cVAC were mainly based on electron microscopy and immunofluorescence tools, and in the previous decade, they have expanded to the use of new technologies and systems approaches (i.e., transcriptomics, proteomics, lipidomics), which led to the acquisition of a vast amount of data. Despite the immense expansion of knowledge about membranous organelle system, including a large amount of data derived from CMV infected cells, the organization and biogenesis of the cVAC remain unclear. Accordingly, very little is known about the processes of a final stage of CMV maturation and the virion egress. This minireview summarizes current knowledge about the biogenesis of the betaherpesvirus factory from the already existent endosomal compartments

Quantitative analysis of endocytic recycling of membrane proteins by monoclonal antibody-based recycling assays

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols

Cytoplasmic virion assembly compartment of betaherpesviruses

During their life cycle, betaherpesviruses extensively reorganize the membranous system of the cell in order to develop a unique cytoplasmic environment of virion production ("virus factory"), known as cytoplasmic virion assembly compartment (cVAC). The betaherpesvirus cVAC has been studied both in human and murine cytomegalovirus (CMV) infected cells as they are considered to be models for betaherpesviruses biology. Studies of the cVAC were mainly based on electron microscopy and immunofluorescence tools, and in the previous decade, they have expanded to the use of new technologies and systems approaches (i.e., transcriptomics, proteomics, lipidomics), which led to the acquisition of a vast amount of data. Despite the immense expansion of knowledge about membranous organelle system, including a large amount of data derived from CMV infected cells, the organization and biogenesis of the cVAC remain unclear. Accordingly, very little is known about the processes of a final stage of CMV maturation and the virion egress. This minireview summarizes current knowledge about the biogenesis of the betaherpesvirus factory from the already existent endosomal compartments

Biofilm Degradation of Nontuberculous Mycobacteria Formed on Stainless Steel Following Treatment with Immortelle (Helichrysum italicum) and Common Juniper (Juniperus communis) Essential Oils

Nontuberculous mycobacteria, like other opportunistic premise plumbing pathogens, produce resistant biofilms on various surfaces in the plumbing system including pipes, tanks, and fittings. Since standard methods of water disinfection are ineffective in eradicating biofilms, research into new agents is necessary. Essential oils (EOs) have great potential as anti-biofilm agents. Therefore, the purpose of this research was to investigate the potential anti-biofilm effect of common juniper (Juniperus communis) and immortelle (Helichrysum italicum) EOs. Minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), and minimum effective concentrations of EOs on Mycobacterium avium, M. intracellulare, and M. gordonae were tested. Additionally, biofilms on the surface of a stainless steel disc were treated with single or mixed concentration of EOs, in order to investigate their degeneration via the bacterial count and confocal laser scanning microscopy (CLSM). H. italicum EO showed the strongest biofilm degradation ability against all Mycobacteria strains that were tested. The strongest effect in the biofilm degradation after the single or mixed applications of EOs was observed against M. gordonae, followed by M. avium. The most resistant was the M. intracellulare biofilm. Synergistic combinations of J. communis and H. italicum EOs therefore seem to be an effective substance in biofilm degradation for use in small water systems such as baths or hot tubs