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Article ; Online: Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods

Veronica Di Antonio / Giorgio Palù / Gualtiero Alvisi

Microorganisms, Vol 9, Iss 928, p

2021 Volume 928

Abstract: Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication ... ...

Abstract	Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRET max of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.
Keywords	DNA polymerase ; BRET ; qBRET ; UL44 ; ppUL44 ; UL54 ; Biology (General) ; QH301-705.5
Subject code	570 ; 612
Language	English
Publishing date	2021-04-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Why Cells and Viruses Cannot Survive without an ESCRT

Arianna Calistri / Alberto Reale / Giorgio Palù / Cristina Parolin

Cells, Vol 10, Iss 483, p

2021 Volume 483

Abstract: Intracellular organelles enwrapped in membranes along with a complex network of vesicles trafficking in, out and inside the cellular environment are one of the main features of eukaryotic cells. Given their central role in cell life, compartmentalization ...

Abstract	Intracellular organelles enwrapped in membranes along with a complex network of vesicles trafficking in, out and inside the cellular environment are one of the main features of eukaryotic cells. Given their central role in cell life, compartmentalization and mechanisms allowing their maintenance despite continuous crosstalk among different organelles have been deeply investigated over the past years. Here, we review the multiple functions exerted by the endosomal sorting complex required for transport (ESCRT) machinery in driving membrane remodeling and fission, as well as in repairing physiological and pathological membrane damages. In this way, ESCRT machinery enables different fundamental cellular processes, such as cell cytokinesis, biogenesis of organelles and vesicles, maintenance of nuclear–cytoplasmic compartmentalization, endolysosomal activity. Furthermore, we discuss some examples of how viruses, as obligate intracellular parasites, have evolved to hijack the ESCRT machinery or part of it to execute/optimize their replication cycle/infection. A special emphasis is given to the herpes simplex virus type 1 (HSV-1) interaction with the ESCRT proteins, considering the peculiarities of this interplay and the need for HSV-1 to cross both the nuclear-cytoplasmic and the cytoplasmic-extracellular environment compartmentalization to egress from infected cells.
Keywords	ESCRT ; viruses ; cellular membranes ; extracellular vesicles ; HSV-1 ; Biology (General) ; QH301-705.5
Subject code	571
Language	English
Publishing date	2021-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Vaccination Criteria based on Factors Influencing COVID-19 Diffusion and Mortality

Ilaria Spassiani / Lorenzo Gubian / Giorgio Palù / Giovanni Sebastiani

Vaccines, Vol 8, Iss 766, p

2020 Volume 766

Abstract: SARS-CoV-2 is highly contagious, rapidly turned into a pandemic, and is causing a relevant number of critical to severe life-threatening COVID-19 patients. However, robust statistical studies of a large cohort of patients, potentially useful to implement ...

Abstract	SARS-CoV-2 is highly contagious, rapidly turned into a pandemic, and is causing a relevant number of critical to severe life-threatening COVID-19 patients. However, robust statistical studies of a large cohort of patients, potentially useful to implement a vaccination campaign, are rare. We analyzed public data of about 19,000 patients for the period 28 February to 15 May 2020 by several mathematical methods. Precisely, we describe the COVID-19 evolution of a number of variables that include age, gender, patient’s care location, and comorbidities. It prompts consideration of special preventive and therapeutic measures for subjects more prone to developing life-threatening conditions while affording quantitative parameters for predicting the effects of an outburst of the pandemic on public health structures and facilities adopted in response. We propose a mathematical way to use these results as a powerful tool to face the pandemic and implement a mass vaccination campaign. This is done by means of priority criteria based on the influence of the considered variables on the probability of both death and infection.
Keywords	SARS-CoV-2 ; statistical analysis ; vaccines ; pandemic preparedness ; Medicine ; R
Subject code	310
Language	English
Publishing date	2020-12-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Human Monocytes Are Suitable Carriers for the Delivery of Oncolytic Herpes Simplex Virus Type 1 In Vitro and in a Chicken Embryo Chorioallantoic Membrane Model of Cancer

Alberto Reale / Lea Krutzke / Massimiliano Cadamuro / Adriana Vitiello / Jens von Einem / Stefan Kochanek / Giorgio Palù / Cristina Parolin / Arianna Calistri

International Journal of Molecular Sciences, Vol 24, Iss 9255, p

2023 Volume 9255

Abstract: Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the ... ...

Abstract	Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models.
Keywords	oncolytic virus ; virotherapy ; HSV-1 ; monocytes ; carrier cells ; CAM model ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	610
Language	English
Publishing date	2023-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Conserved presence of G-quadruplex forming sequences in the Long Terminal Repeat Promoter of Lentiviruses

Rosalba Perrone / Enrico Lavezzo / Giorgio Palù / Sara N. Richter

Scientific Reports, Vol 7, Iss 1, Pp 1-

2017 Volume 11

Abstract: Abstract G-quadruplexes (G4s) are secondary structures of nucleic acids that epigenetically regulate cellular processes. In the human immunodeficiency lentivirus 1 (HIV-1), dynamic G4s are located in the unique viral LTR promoter. Folding of HIV-1 LTR ... ...

Abstract	Abstract G-quadruplexes (G4s) are secondary structures of nucleic acids that epigenetically regulate cellular processes. In the human immunodeficiency lentivirus 1 (HIV-1), dynamic G4s are located in the unique viral LTR promoter. Folding of HIV-1 LTR G4s inhibits viral transcription; stabilization by G4 ligands intensifies this effect. Cellular proteins modulate viral transcription by inducing/unfolding LTR G4s. We here expanded our investigation on the presence of LTR G4s to all lentiviruses. G4s in the 5′-LTR U3 region were completely conserved in primate lentiviruses. A G4 was also present in a cattle-infecting lentivirus. All other non-primate lentiviruses displayed hints of less stable G4s. In primate lentiviruses, the possibility to fold into G4s was highly conserved among strains. LTR G4 sequences were very similar among phylogenetically related primate viruses, while they increasingly differed in viruses that diverged early from a common ancestor. A strong correlation between primate lentivirus LTR G4s and Sp1/NFκB binding sites was found. All LTR G4s folded: their complexity was assessed by polymerase stop assay. Our data support a role of the lentiviruses 5′-LTR G4 region as control centre of viral transcription, where folding/unfolding of G4s and multiple recruitment of factors based on both sequence and structure may take place.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2017-05-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells

Lorenzo Messa / Marta Celegato / Chiara Bertagnin / Beatrice Mercorelli / Giulio Nannetti / Giorgio Palù / Arianna Loregian

Scientific Reports, Vol 8, Iss 1, Pp 1-

2018 Volume 11

Abstract: Abstract High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of ... ...

Abstract	Abstract High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2018-04-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells

Marta Trevisan / Giovanna Desole / Giulia Costanzi / Enrico Lavezzo / Giorgio Palù / Luisa Barzon

International Journal of Molecular Sciences, Vol 18, Iss 1, p

2017 Volume 206

Abstract: Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to ... ...

Abstract	Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.
Keywords	reprogramming method ; induced pluripotent stem cells ; retroviral vector ; Sendai virus vector ; episomal vector ; gene expression ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	616
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Publisher Correction

Francesca Boldrin / Laura Cioetto Mazzabò / Saber Anoosheh / Giorgio Palù / Luc Gaudreau / Riccardo Manganelli / Roberta Provvedi

Scientific Reports, Vol 9, Iss 1, Pp 1-

Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE

2019 Volume 1

Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper. ...

Abstract	An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2019-11-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system

Francesca Boldrin / Saber Anoosheh / Agnese Serafini / Laura Cioetto Mazzabò / Giorgio Palù / Roberta Provvedi / Riccardo Manganelli

Scientific Reports, Vol 9, Iss 1, Pp 1-

2019 Volume 6

Abstract: Abstract Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for ... ...

Abstract	Abstract Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo. One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants.
Keywords	Medicine ; R ; Science ; Q
Subject code	572 ; 570
Language	English
Publishing date	2019-04-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE

Francesca Boldrin / Laura Cioetto Mazzabò / Saber Anoosheh / Giorgio Palù / Luc Gaudreau / Riccardo Manganelli / Roberta Provvedi

Scientific Reports, Vol 9, Iss 1, Pp 1-

2019 Volume 7

Abstract: Abstract σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins ...

Abstract	Abstract σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor.
Keywords	Medicine ; R ; Science ; Q
Subject code	306
Language	English
Publishing date	2019-03-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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