Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus

Replication 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

Human CMV Polymerase Holoenzyme Nuclear Import: Regulation by PKC Phosphorylation of Processivity Factor ppUL44

Replication of the human cytomegalovirus (HCMV) 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, which tethers the catalytic subunit to DNA. ppUL44 dimerizes in the cytoplasm of infected cells before being translocated to the nucleus and has been proposed to act as a scaffold promoting the assembly of several HCMV replication fork proteins, such as pUL54 and the DNA uracil glycosylase pUL114. Although both pUL44 and pUL54 contain importin \u3b1/\u3b2 recognized nuclear localization signals (NLSs) they are also capable of being imported into the nucleus as a complex. Whereas ppUL44 nuclear import is a CK2-phosphorylation enhanced process, pUL54\u2019s is not. Here we show that ppUL44 nuclear import is likely to be also negatively regulated by protein kinase C-mediated phosphorylation of residue T427. As shown by quantitative confocal laser scanning microscopic analysis of live COS-7 cells expressing several ppUL44 GFP and DsRed2 fusion proteins, Phorbol 12-myristate 13-acetate (PMA)-induced PKC activation resulted in reduction of the nuclear accumulation of GFP-UL44 but not of the non phosphorylable A427 derivative mutant. In the absence of PMA, the phosphomimetic D427 derivative mutant exhibited reduced nuclear import when compared to the wild-type and the A427 mutant. Since ppUL114 nuclear accumulation seems to be dependent on ppUL44, we suggest that the phosphorylation state of ppUL44 could regulate the nuclear import rate of the HCMV DNA polymerase holoenzyme, and other viral proteins such as ppUL114

Gli oli essenziali di Monarda fistulosa e Monarda didyma inibiscono l\u2019infettivit\ue0 dei virus Herpes simplex I e II in vitro.

I virus Herpes simplex (HSV) 1 e 2, comunemente noti come herpes labiale e genitale, sono molto diffusi nella popolazione umana e la loro sieroprevalenza pu\uf2 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\u2019encefalo. Pi\uf9 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\u2019infezione ma non portano all\u2019eradicazione dei virus. Inoltre il loro ampio uso favorisce l\u2019insorgenza di ceppi resistenti, in particolare in pazienti immunocompromessi. E\u2019 dunque importante individuare nuovi approcci di profilassi e/o terapia con l\u2019intento di affiancare alle molecole in uso composti con meccanismi d\u2019azione alternativi. Scopo. Valutare la capacit\ue0 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 infettivit\ue0, 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 attivit\ue0 antivirale sono stati effettuati nelle seguenti condizioni: (a) preincubazione virus/OE, (b) preincubazione monostrati cellulari con /OE, (c) aggiunta degli OE successiva all\u2019adsorbimento 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\u2019adsorbimento 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, pi\uf9 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\u2019infezione e contenere la diffusione del virus. La disponibilit\ue0 di molecole in grado di agire su fasi distinte del ciclo replicativo virale offre la possibilit\ue0 di ridurre le dosi dei farmaci utilizzati di concerto e la durata del trattamento, minimizzando i rischi d\u2019insorgenza di virus resistenti e di effetti indesiderati

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

Although 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

Regulated nucleocytoplasmic trafficking of viral gene products: A therapeutic target?

The study of viral proteins and host cell factors that interact with them has represented an invaluable contribution to understanding of the physiology as well as associated pathology of key eukaryotic cell processes such as cell cycle regulation, signal transduction and transformation. Similarly, knowledge of nucleocytoplasmic transport is based largely on pioneering studies performed on viral proteins that enabled the first sequences responsible for the facilitated transport through the nuclear pore to be identified. The study of viral proteins has also enabled the discovery of several nucleocytoplasmic regulatory mechanisms, the best characterized being through phosphorylation. Recent delineation of the mechanisms whereby phosphorylation regulates nuclear import and export of key viral gene products encoded by important human pathogens such as human cytomegalovirus dengue virus and respiratory syncytial virus has implications for the development of antiviral therapeutics. In particular, the development of specific and effective kinase inhibitors makes the idea of blocking viral infection by inhibiting the phosphorylation-dependent regulation of viral gene product nuclear transport a real possibility. Additionally, examination of a chicken anemia virus (CAV) protein able to target selectively into the nucleus of tumor but not normal cells, as specifically regulated by phosphorylation, opens the exciting possibility of cancer cell-specific nuclear targeting. The study of nucleoplasmic transport may thus enable the development not only of new antiviral approaches, but also contribute to anti-cancer strategies