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Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus
Full text linkReplication of the human cytomegalovirus genome takes place in the nuclei of infected cells and is mediated by a viral-encoded DNA polymerase complex formed by the catalytic subunit pUL54 and the processivity factor ppUL44. Although it has recently been shown that the dimerization ability of recombinant pUL44 appears to be crucial for effective DNA binding in vitro, whether ppUL44 can dimerize or not in a cellular context is unknown. Here, we show, by using co-immunoprecipitation and dual-color live imaging approaches on cells expressing fluorescent and differently tagged ppUL44 fusion proteins, that ppUL44 dimerizes in the cytoplasm via its N-terminal domain, before translocating to the nucleus. Furthermore, we show that nuclear translocation of differently tagged ppUL44 heterodimers can occur even when one subunit carries a nonfunctional nuclear localization signal. Importantly, the latter cotransfection assay represents a system to test small-molecule inhibitors for their ability to impair ppUL44 dimerization
Gli oli essenziali di Monarda fistulosa e Monarda didyma inibiscono l’infettività dei virus Herpes simplex I e II in vitro.
No full textI virus Herpes simplex (HSV) 1 e 2, comunemente noti come herpes labiale e genitale, sono molto diffusi nella popolazione umana e la loro sieroprevalenza può arrivare fino al 90% in popolazioni sfavorite dal punto di vista socioeconomico. Le infezioni da HSV possono decorrere asintomatiche o essere gravissime, per esempio in ambito perinatale, nel soggetto immunodepresso, o quando coinvolgono l’encefalo. Più comunemente provocano lesioni dolorose e ricorrenti con un forte impatto negativo sulla vita di relazione dei pazienti. Non si dispone di un vaccino contro HSV, mentre i farmaci disponibili possono controllare le fasi acute dell’infezione ma non portano all’eradicazione dei virus. Inoltre il loro ampio uso favorisce l’insorgenza di ceppi resistenti, in particolare in pazienti immunocompromessi.
E’ dunque importante individuare nuovi approcci di profilassi e/o terapia con l’intento di affiancare alle molecole in uso composti con meccanismi d’azione alternativi.
Scopo. Valutare la capacità degli olii essenziali (OE) estratti da Monarda spp. di contrastare il ciclo vitale dei virus HSV-1 e HSV-2.
Materiali e metodi. Cellule: abbiamo utilizzato la linea cellulare di astrocitoma U-373 MG per tutti gli esperimenti di infettività, mentre le line Vero (per HSV-1) e BHK (per HSV-2) sono state impiegate per la titolazione dei virus. I virus utilizzati sono stati un isolato clinico di HSV-1, il mutante replicazione competente K26GFP, che esprime la proteina fluorescente GFP e il ceppo di laboratorio HSV-2 (G). Infine sono stati impiegati OE di Monarda fistulosa, M. didyma e Melaleuca alternifolia. Saggi di attività antivirale sono stati effettuati nelle seguenti condizioni: (a) preincubazione virus/OE, (b) preincubazione monostrati cellulari con /OE, (c) aggiunta degli OE successiva all’adsorbimento virale su cellule preincubate con gli olii, (d) aggiunta degli OE dopo adsorbimento con virus preincubato con gli olii, (e) aggiunta degli OE subito dopo l’adsorbimento di virus. Le cellule venivano fissate ai tempi 3, 6, 9, 12, 18, 24 e 48 ore post infectionem. Il grado di replicazione virale veniva giudicato in confronto a cellule non trattate infettate con virus non trattato, osservando la comparsa di proteine virus specifiche o contando le placche da effetto citopatico.
Risultati. Abbiamo osservato un forte effetto virucida degli OE delle due specie di Monarda su HSV-1 e 2, più potente di quello noto per M. alternifolia. Gli OE non sono invece in grado di contrastare la replicazione virale una volta che i virus siano penetrati nelle cellule.
Conclusioni. Il potere virucida di questi OE si pone come un promettente presidio per prevenire l’infezione e contenere la diffusione del virus. La disponibilità di molecole in grado di agire su fasi distinte del ciclo replicativo virale offre la possibilità di ridurre le dosi dei farmaci utilizzati di concerto e la durata del trattamento, minimizzando i rischi d’insorgenza di virus resistenti e di effetti indesiderati
A protein kinase CK2 site flanking the nuclear targeting signal enhances nuclear transport of human cytomegalovirus ppUL44
Full text linkThe processivity factor of the human cytomegalovirus (HCMV) DNA polymerase phosphoprotein ppUL44 plays an essential role in viral replication, showing nuclear localization in infected cells. The present study examines ppUL44's nuclear import pathway for the first time, ectopic expression of ppUL44 revealing a strong nuclear localization in transfected COS-7 and other cell types, implying that no other HCMV proteins are required for nuclear transportation and retention. We show that of the two potential nuclear localization signals (NLSs) located at amino acids 162-168 (NLS1) and 425-431 (NLS2), NLS2 is necessary and sufficient to confer nuclear localization. Moreover, using enzyme-linked immunosorbent assays and gel mobility shift assays, we show that NLS2 is recognized with high affinity by the importin (IMP) alpha/beta heterodimer. Using gel mobility shift and transient transfection assays, we find that flanking sequences containing a cluster of potential phosphorylation sites, including a consensus site for protein kinase CK2 (CK2) at Ser413 upstream of the NLS, increase NLS2-dependent IMP binding and nuclear localization, suggesting a role for these sites in enhancing UL44 nuclear transport. Results from site-directed mutagenic analysis and live-cell imaging of green fluorescent protein (GFP)-UL44 fusion protein-expressing cells treated with the CK2-specific inhibitor 4,5,6,7-tetrabromobenzotriazole are consistent with phosphorylation of Ser413 enhancing ppUL44 nuclear transport
An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus
Full text linkAlthough the 1235 amino acids human herpes simplex virus type 1 (HSV-1) DNA polymerase catalytic subunit, pUL30, is essential for HSV-1 replication in the nucleus of host cells, little information is available regarding its nuclear import mechanism. The present study addresses this issue directly, characterizing pUL30's nuclear import pathway for the first time using quantitative confocal laser scanning microscopy (CLSM) on living cells, and fluorescent binding assays. In addition to a previously described nuclear localization signal (NLS) located within the pUL30 binding site for the polymerase accessory protein (PAP) pUL42, that appears to be dispensable for nuclear targeting, pUL30 possesses three putative basic NLSs. Intriguingly, the core of pUL30-NLS2 (residues 1114-1120) is highly homologous to that of the recently described NLS, similarly located upstream of the PAP binding site, of the human cytomegalovirus (HCMV) DNA polymerase catalytic subunit, pUL54. Here we show for the first time that pUL30-NLS2 itself is only partially functional in terms of nuclear import due to residue P1118 present in position 3 of the NLS core. Intriguingly, pUL30-NLS2 together with pUL30-NLS3 (residues 1133-1136) represents a fully functional bipartite NLS (pUL30-NLSbip), required for nuclear targeting of pUL30, and able to confer nuclear localization on heterologous proteins by conferring high-affinity interaction with the importin (IMP) alpha/beta heterodimer. Since nuclear targeting of HSV-1 proteins forming the replication fork is crucial for viral replication, the pUL30-NLSbip emerges for the first time as a viable therapeutic target
ANTIBODY RESPONSE TO INDIVIDUAL CYTOMEGALOVIRUS STRUCTURAL PROTEINS IN DIFFERENT GROUPS OF SUBJECTS
No full textHuman cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs
Full text linkThe catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import
A novel Western blot test containing both viral and recombinant proteins for anticytomegalovirus immunoglobulin M detection.
No full textWe devised a novel Western blot (WB) test for anti-human cytomegalovirus (HCMV) immunoglobulin M (IgM) detection which contains viral structural polypeptides, significant portions of recombinant p150 (ppUL32), and a significant portion of the must immunogenic nonstructural protein p52 (ppUL44). This new test was evaluated in latently infected blood donors, pregnant women, and transplant recipients with ongoing HCMV infection and shown to be more sensitive and specific than traditional WB and conventional enzyme immunoassay for the detection of HCMV-specific IgM
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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