Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

We have previously shown that clusters of guanine quadruplex (G4) structures can form in the human herpes simplex-1 (HSV-1) genome. Here we used immunofluorescence and immune-electron microscopy with a G4-specific monoclonal antibody to visualize G4 structures in HSV-1 infected cells. We found that G4 formation and localization within the cells was virus cycle dependent: viral G4s peaked at the time of viral DNA replication in the cell nucleus, moved to the nuclear membrane at the time of virus nuclear egress and were later found in HSV-1 immature virions released from the cell nucleus. Colocalization of G4s with ICP8, a viral DNA processing protein, was observed in viral replication compartments. G4s were lost upon treatment with DNAse and inhibitors of HSV-1 DNA replication. The notable increase in G4s upon HSV-1 infection suggests a key role of these structures in the HSV-1 biology and indicates new targets to control both the lytic and latent infection

G-quadruplexes in the HSV-1 and HHV-6 genomes as antiviral targets

Guanine-rich nucleic acids can fold into G-quadruplexes, four-stranded secondary structures which are implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. Because the herpes simplex virus-1 (HSV-1) genome is remarkably rich in guanines, we aimed at investigating both the presence of G-quadruplex forming sequences at the viral genome level and the possibility to target them with G-quadruplex ligands to obtain anti-HSV-1 effects with novel mechanisms of action. Here we show that HSV-1 displays six clusters of repeated sequences that form very stable G-quadruplexes. These sequences are located in the inverted repeats and in two gene-coding regions (ICP0 and UL36) of the HSV-1 genome. One G-quadruplex repeat is located in the promoter region of the multifunctional protein γ134.5. Treatment of HSV-1 infected cells with the G-quadruplex ligands BRACO-19 and TMPyP4 induced significant inhibition of virus production and reduction of viral transcripts. BRACO-19 was able to inhibit Taq polymerase processing at G-quadruplex forming sequences in the HSV-1 genome, and caused a decreased intracellular viral DNA in infected cells. The last step targeted by BRACO-19 was viral DNA replication, while no effect on virus entry in the cells was observed. A different TMPyP4-mediated mechanism of action was on the contrary observed. Despite its capability to affect Taq polymerase processing, TMPyP4 did not inhibit intracellular viral DNA and it appeared to prevent HSV-1 maturation/egress by stimulating the autophagy process. As a second part of the study, we extended this innovative antiviral approach to human herpes virus-6 (HHV-6). One of the main HHV-6 features is the presence of tandem repeats of the telomeric sequence (TTAGGG)n at the genome termini (DR). This peculiarity is thought to be responsible for the viral integration in specific human chromosomes, occurring in the 1-2% of the world population. To date, the telomeric G-quadruplex structure had been extensively characterized. We showed that BRACO-19 and TMPyP4 displayed a great antiviral activity against both HHV-6A and HHV-6B. In the third part of this study, by using specific DNA G-quadruplex-interacting antibodies, for the first time we visualized viral DNA G-quadruplexes in infected cells at crucial time points for the viral replication cycle, in which viral DNA is likely in a single-stranded state. This work, besides presenting the first evidence of extended G-quadruplex sites in key regions of the HSV-1 and HHV-6 genomes, points out G-quadruplexes as innovative potential antiviral targets in novel therapeutic interventions, based on the use of G-quadruplex ligands

Levodopa-Induced Dyskinesias and Dyskinesias-Reduced-Self-Awareness in Parkinson’s Disease: A Neurocognitive Approach

Levodopa-induced dyskinesias are one of the most common disabling motor complications in advanced Parkinson’s disease. The subjective perception of motor impairment is a clinical phenomenon that needs to be adequately analyzed. Indeed, the determination of patient dyskinesias-reduced-self-awareness (DRSA) and of its relationship to daily dysfunction is an important aspect of the debate on the gold standard for treatment. As the association with executive dysfunction is a matter of debate and we hypothesize it plays an important role in DRSA, we analyzed metacognitive abilities related to action monitoring and other factors, such as response-inhibition and “Theory of Mind,” which represent a novel explanation of the phenomenon. Moreover, we investigated whether and how a dysfunction in action monitoring related to the cingulo-frontal-ventral striatal circuit would be associated with DRSA using an event-related Go-NoGo fMRI experiment. Our findings suggest the presence of executive dysfunctions in DRSA pathogenesis, with a key leading role played by the cingulo-frontal network as part of a functionally impaired response-inhibition network

Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways

Pisa Syndrome in Parkinson's Disease Is Associated With Specific Cognitive Alterations

Background: Pisa syndrome (PS) is a lateral flexion of the trunk frequently associated with Parkinson's disease (PD). The pathophysiology of PS remains unclear, but the role of cognitive deficits has been postulated.Methods: We included 12 consecutive PD patients with PS (PS+) and 12 PD patients without PS (PS–) matched for gender, age, level of education, PD duration, and PD stage. As primary aim, we compared the neuropsychological scores of 16 tests evaluating 6 cognitive domains between PS+ and PS–. Additionally, we evaluated the presence of misperception of the trunk position in PS+, defined as a mismatch between the objective vs. subjective evaluation of the trunk bending angle >5°, and analyzed whether a correlation exists between the misperception of the trunk position and alterations in the visual-spatial abilities.Results: PS+ group showed significantly worse performances in the visual-spatial abilities (p: 0.008), attentional domain (p: 0.001), and language domain (p: 0.023). No differences were found in the other cognitive domains nor in the general cognitive assessment. All PS+ patients showed a misperception of the trunk position, with an average underestimation of the trunk bending angle of 11.7° ± 4.3. The degree of misperception of the trunk position showed a trend toward a correlation with the visual-spatial scores (p: 0.089).Conclusions: The study reveals an association between PS and specific cognitive alterations, suggesting a possible link between the abnormal posture of PD patients with PS and their cognitive functions

O31 Integrative analysis reveals a molecular stratification of systemic autoimmune diseases

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G-quadruplexes in the HSV-1 and HHV-6 genomes as antiviral targets

Guanine-rich nucleic acids can fold into G-quadruplexes, four-stranded secondary structures which are implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. Because the herpes simplex virus-1 (HSV-1) genome is remarkably rich in guanines, we aimed at investigating both the presence of G-quadruplex forming sequences at the viral genome level and the possibility to target them with G-quadruplex ligands to obtain anti-HSV-1 effects with novel mechanisms of action. Here we show that HSV-1 displays six clusters of repeated sequences that form very stable G-quadruplexes. These sequences are located in the inverted repeats and in two gene-coding regions (ICP0 and UL36) of the HSV-1 genome. One G-quadruplex repeat is located in the promoter region of the multifunctional protein γ134.5. Treatment of HSV-1 infected cells with the G-quadruplex ligands BRACO-19 and TMPyP4 induced significant inhibition of virus production and reduction of viral transcripts. BRACO-19 was able to inhibit Taq polymerase processing at G-quadruplex forming sequences in the HSV-1 genome, and caused a decreased intracellular viral DNA in infected cells. The last step targeted by BRACO-19 was viral DNA replication, while no effect on virus entry in the cells was observed. A different TMPyP4-mediated mechanism of action was on the contrary observed. Despite its capability to affect Taq polymerase processing, TMPyP4 did not inhibit intracellular viral DNA and it appeared to prevent HSV-1 maturation/egress by stimulating the autophagy process. As a second part of the study, we extended this innovative antiviral approach to human herpes virus-6 (HHV-6). One of the main HHV-6 features is the presence of tandem repeats of the telomeric sequence (TTAGGG)n at the genome termini (DR). This peculiarity is thought to be responsible for the viral integration in specific human chromosomes, occurring in the 1-2% of the world population. To date, the telomeric G-quadruplex structure had been extensively characterized. We showed that BRACO-19 and TMPyP4 displayed a great antiviral activity against both HHV-6A and HHV-6B. In the third part of this study, by using specific DNA G-quadruplex-interacting antibodies, for the first time we visualized viral DNA G-quadruplexes in infected cells at crucial time points for the viral replication cycle, in which viral DNA is likely in a single-stranded state. This work, besides presenting the first evidence of extended G-quadruplex sites in key regions of the HSV-1 and HHV-6 genomes, points out G-quadruplexes as innovative potential antiviral targets in novel therapeutic interventions, based on the use of G-quadruplex ligands.Acidi nucleici ricchi di guanine possono formare una particolare struttura secondaria nota come G-quadruplex, il quale è implicato nella regolazione di importanti processi biologici a livello del genoma umano, di procarioti e virus. Dato che il genoma dell’herpes simplex virus-1 (HSV-1) è notevolmente ricco in guanine, il nostro studio è stato mirato all’individuazione di sequenze virali in grado di foldare in G-quadruplex e, in secondo luogo, alla possibilità di colpire selettivamente questa conformazione non-canonica con specifiche molecole chimiche, al fine di sviluppare una terapia antivirale innovativa. Il nostro studio dimostra la presenza di sei gruppi composti da sequenze altamente ripetute capaci di formare strutture G-quadruplex particolarmente stabili. Queste sequenze sono state individuate a livello delle regioni terminali del genoma di HSV-1 e all’interno di due regioni codificanti (geni ICP0 e UL36). Un cluster di sequenze G-quadruplex, inoltre, è situato nella regione promotoriale deputata all’espressione della proteina multifunzionale γ134.5. Il trattamento di cellule infettate con BRACO-19 e TMPyP4 ha mostrato una significativa inibizione di HSV-1 e una riduzione dell’espressione di alcuni trascritti virali. BRACO-19 si è dimostrato efficace nell’inibire l’elongazione di regioni G-quadruplex del genoma di HSV-1, da parte dell’enzima Taq; è stato in grado di diminuire il DNA virale intracellulare, opportunamente estratto da cellule infettate e trattate; inoltre, mentre nessun effetto è stato osservato sull’entry di HSV-1, l’ultimo step del ciclo virale inibito da BRACO-19 corrisponde alla replicazione del DNA virale. Un diverso meccanismo d’azione è stato, invece, osservato in seguito al trattamento con TMPyP4. Nonostante questa porfirina sia riuscita ad inibire l’attività della Taq, non è riuscita, al contrario, ad influenzare i livelli di DNA virali intracellulari. TMPyP4 sembra essere efficace nell’inibire la maturazione/uscita di HSV-1, attraverso un’induzione dell’autofagia. In una seconda parte dello studio, abbiamo esteso questa promettente strategia antivirale all’herpes virus umano 6 (HHV-6), il quale presenta una caratteristica molto peculiare. Ad entrambe le estremità del suo genoma (DR) contiene, infatti, numerose ripetizioni della sequenza telomerica umana (TTAGGG)n. Questa caratteristica è ritenuta responsabile dell’integrazione di HHV-6 in alcuni cromosomi umani (1-2% della popolazione). Ad oggi, la struttura G-quadruplex della sequenza telomerica è stata studiata in dettaglio e completamente caratterizzata. Abbiamo, quindi, testato BRACO-19 e TMPyP4 su cellule infettate sia con HHV-6A che HHV-6B, ottenendo anche in questo caso dei risultati molto incoraggianti. Infine, in una terza parte del lavoro, è stato possibile visualizzare per la prima volta G-quadruplex virali a livello del DNA in cellule infettate, grazie all’impiego di anticorpi ad alta affinità per questa struttura. La specificità di segnale è data dal fatto che l’incremento di fluorescenza è avvenuto nel corso della replicazione virale in cui il DNA è presente come singolo filamento. Questo lavoro, oltre a mostrare l’esistenza di estese regioni G-quadruplex in siti chiave del genoma di HSV-1 e HHV-6, mette in luce le potenzialità del G-quadruplex come target innovativo in terapie antivirali nuove e mirate, basate sull’impiego di leganti specifici per questa particolare conformazione secondaria del DNA

Struttura G-quadruplex nel genoma di HSV-1: studi in vitro e in linee cellulari

E' stata condotta un'analisi computazionale sul genoma di HSV-1, per ricercare sequenze ricche in guanina capaci di ripiegarsi a G-quadruplex. E' stato dimostrato il folding a G-quadruplex in vitro, l'interazione con i ligandi TMPyP4, BRACO-19, PIPER e Telomestatina. Studi di titolazione virale, condotti usando il ceppo F di HSV-1 hanno portato ad osservare diminuzione virale con TMPyP4