Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2-P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2-5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2-3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

Corneal diseases are among the most prevalent causes of blindness worldwide. The transparency and clarity of the cornea are guaranteed by a delicate physiological, anatomic and functional balance. For this reason, all the disorders, including those of genetic origin, which compromise this state of harmony can lead to opacity and eventually vision loss. Many corneal disorders have a genetic etiology and some are associated with rather rare and complex syndromes. Conventional treatments, such as corneal transplantation, are often ineffective and, to date, many of these disorders are still incurable. Gene therapy carries the promise to be a potential cure for many of these diseases, with solutions and strategies that did not seem possible until a few years ago.With its potential to treat genetic disease by means of deletion, replacement or editing of a defective gene, the challenge can also be extended to corneal disorders in order to achieve long-term, if not definitive, relief.The aim of this paper is to review the state of the art of the different gene therapy approaches as potential treatments for corneal diseases and the future perspectives for the development of personalized gene-based medicine.

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited HCMV AD169 strain in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

16Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised 17 individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite 18 the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and 19 resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. 20 The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the 21 viral life cycle being required for oriLyt-dependent DNA replication. We validated the existence of 22 UL44 homodimers both in vitro and in living cells by a variety of approaches, including GST 23 pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable 24 to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the 25 dimerization interface. Subsequently, we performed an in-silico screening to select 18 small 26 molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using 27 recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification 28 of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and 29 impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a 30 similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically 31 reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA 32 synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV 33 inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase 34 complex. 35 IMPORTANCE 36 HCMV is a ubiquitous infectious agent causing life-lasting infections in humans. HCMV primary 37 infections and reactivation in non-immunocompetent individuals often result in life-threatening 38 conditions. Antiviral therapy mainly targets the DNA polymerase catalytic subunit UL54 and is often 39 limited by toxicity and selection of drug-resistant viral strains, making the identification of new 40 targets of therapeutic intervention crucial for a successful management of HCMV infections. The 41 49The b-Herpesvirinae member human cytomegalovirus (HCMV) is a major human pathogen, causing 50 severe and life-threatening infections in immunocompromised patients (1) and in congenitally 51 infected newborns (2). Herpesviruses are opportunistic double-stranded DNA viruses, whose genome 52 transcription, replication, and packaging occur in the host cell nucleus (3). The molecular mechanisms 53 involved in herpesvirus DNA replication and its regulation have been widely studied as they provide 54 important models for the study of eukaryotic DNA replication and because viral enzymes involved 55 in the process represent targets for antiviral therapy. HCMV DNA polymerase holoenzyme is a multi-56 functional enzyme that play...