No description available.

Cytomegalovirus (CMV) infections remain a significant problem in congenitally infected infants and immunocompromised individuals. Modest antiviral activities of currently approved drugs coupled with dose-limiting toxicities restrict effectiveness and promote development of resistance. The potential for ribonucleotide reductase (RR) inhibitors hydroxyurea (HU), Didox, and Trimidox to synergize, through reduction of nucleotide pools, with the deoxynucleotide analog Ganciclovir (GCV) was examined. A yield reduction assay that utilizes luciferase expressed by a recombinant virus as a surrogate measure of viral infectious units was developed and used to determine effective dose ranges for each drug. RR inhibitors exhibited intrinsic anti-CMV activities on their own with IC50 values well below toxic levels. Moreover, RR inhibitors significantly synergized with GCV. These findings provide a rationale for exploration of RR inhibitors and deoxynucleotide analogs in anti-CMV combination therapy.

The development of specific antiviral drugs directly targeting the hepatitis C virus (HCV) is clinically important, as the current standard interferon/ribavirin combination treatment is only partially effective, expensive and often associated with severe side effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternative or additional therapy. To date, the development and in vitro evaluation of PIs is restricted to the genotype 1/2 based replicon and the genotype 2a full length viral cell culture system. However, proteases of the different HCV genotypes vary substantially in their amino acid sequence and secondary structure and require separate evaluation of their efficacy before they go into clinical trials. To address this issue, a panel of intra- and intergenotypic recombinants based on the recombinant infectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. The viability of these recombinants was assessed in the Huh7.5 cell culture system, where replicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinants containing genotype 1a, 1b, 3a, 4a and 6a derived proteases were replication defective, whereas the recombinant with genotype 5a derived protease replicated efficiently after acquiring cell culture adaptive mutations. The replacement of not only the NS3 protease gene region, but also its cofactor NS4A, allowed the generation of replication competent intra- and intergenotypic recombinants for all 6 major genotypes. Replacing the NS3 protease of the recombinants with that of patientderived proteases also generated replicating recombinants, greatly expanding the panel of intergenotypic recombinants available for phenotyping and PI evaluation. However, intra- and intergenotypic recombinants showed substantial differences in their replication kinetics, which may be influenced by naturally occurring polymorphism between genotypes and the differential requirement of adaptive/attenuating cell culture mutations. Genotype 1a recombinants replicated very poorly, which may be due to incompatibilities between the type 1a NS3/4A protease and the type 2a backbone. 50% inhibitory concentrations (IC50) of different PIs were measured using Foci Forming Units/ml (FFU/ml) reductions and replication inhibition assays. The different recombinants showed consistent, genotype-associated differences in their susceptibility to the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showing approximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants. These observations are consistent with major differences in response rates found in recent treatment trials of genotype 1, 2 and 3 infected patients. Differences in susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derived recombinants being twice as susceptible than genotype 3a, 4a and 5a derived recombinants. Passaging the intra- and intergenotypic recombinants under increasing concentrations of PI allowed the identification of PI resistance mutations. Resistance mutations to BILN 2061 mapped to the previously identified positions 156 and 168 within the NS3 protease, with a great diversity of amino acid substitutions observed within each genotype. Reintroduction of the identified resistance mutations into the original recombinant viruses conferred increased resistance towards BILN 2061 and some mutations also affected replication kinetics of the recombinants. The developed system will be of major value for the phenotypic characterisation of naturally occurring and treatment induced resistance mutations within all 6 major HCV genotypes towards different PIs. This will allow treatment response predictions for newly developed PIs before they enter clinical trials and the development of individually tailored antiviral treatment regimes.

Proper mRNA nuclear export is essential for harmonious growth and maintenance of a cell. An effective weapon influenza virus employs to hijack a host cell is its ability to inhibit such export. Exactly how influenza virus achieves this inhibition is not fully known. Here, we demonstrate that upon infection, influenza virus degrades two nucleopore proteins (Nup98 and Nup96), which play a key role in mRNA nuclear export. Also, a main virulence factor of influenza virus (non-structural protein 1, NS1) binds directly to NXF1 and E1B-AP5, two key constituents of the mRNA export pathway (NXF1/NXT pathway) responsible for exporting bulk (~70%) mRNA from the nucleus. By increasing the expression levels of members of the NXF1/NXT pathway, we were able to reverse NS1-mediated inhibition of gene expression. On the other hand, by decreasing the levels of members of the NXF1/NXT pathway, we demonstrated that host cells become more sensitive to influenza virus infection and produce more viral particles. These results demonstrate undiscovered influenza-mediated host interactions that may be used to medicinally inhibit influenza virus. To this end, high-throughput screens were designed to identify small molecule antagonists of both NS1-mediated inhibition of gene expression and influenza virus-mediated cell death. Seventy-one compounds were identified, and the most potent molecule (named compound #8) was examined further. We found that compound #8 releases influenza virus-mediated mRNA nuclear export blockage and decreased viral replication and viral gene expression. Thus, the bulk mRNA nuclear export machinery is vital to antiviral response, and compound #8 enhances its ability to fight the cytopathic effects of NS1 and influenza virus. In conclusion, our data demonstrate that the mRNA export machinery is disrupted by influenza virus, and that this machinery also facilitates an antiviral function. We have also shown that these two events can be manipulated chemically to attenuate the negative effect of the virus and enhance the positive antiviral effect of the mRNA export machinery, thereby providing a powerful, new strategy against the ever-present, global threat of influenza virus.

Several viruses from different virus families are known to cause ocular infections, e.g. members of the Adenoviridae, Picornaviridae and the Herpesviridae families. These infections are spread by contact and in the case of adenoviruses (Ads) and picornaviruses they are also highly contagious. The ocular infections caused by Ads and picornaviruses are called epidemic keratoconjunctivitis (EKC) and acute hemorrhagic conjunctivitis (AHC), respectively. Historically, EKC is caused mainly by three types of Ads from species D: Ad8, Ad19 and Ad37. The infection is characterized by keratitis and conjunctivitis but also involves pain, edema, lacrimation and blurred vision. AHC is caused mainly by two types of picornaviruses: coxsackievirus A24v (CVA24v) and enterovirus 70 (EV70), and is characterized by pain, redness, excessive tearing, swelling and subconjunctival hemorrhages. In addition, blurred vision, keratitis, malaise, myalgia, fever, headache and upper respiratory tract symptoms can also be experienced. Both infections are problematic in many parts of the world, affecting millions of people every year. Despite the great need, the only treatment available today is supportive treatment; no antiviral drugs are available to combat these common viral infections. Ad37 has previously been reported to use sialic acid (SA) as its cellular receptor. Since there is no antiviral treatment available against EKC we wanted to evaluate the inhibitory effect of SA-based antiviral compounds on Ad37 binding to and infection of ocular cells. We found that multivalent compounds consisting of SA linked to a globular carrier molecule, in this case human serum albumin, efficiently blocked Ad37 binding and infection at low concentrations. Further attempts were then made to improve the effect by chemically modifying SA monosaccharides. However, no enhanced inhibitory effect was accomplished and the conclusion was that the best inhibitors are based on unmodified SA. We next hypothesized that development of efficient SA-based binding inhibitors may require detailed knowledge about the structure of the SA-containing receptor. Using a battery of biological and biochemical experiments, including glycan array, binding and infection assays, X-ray crystallography and surface plasmon resonance (SPR); we identified a specific glycan involved in the binding and infection of Ad37. This glycan turned out to be a branched, di-SA-containing motif corresponding to the glycan motif of the ganglioside GD1a. However, the ganglioside itself did not function as a cellular receptor, as shown by a number of binding and infection assays. Instead, the receptor consisted of one or more glycoproteins that contain the GD1a glycan motif. This glycan docked with both its SAs into the trimeric Ad37 knob resulting in a very strong interaction as compared to most other protein-glycan interactions. Hopefully, this finding will aid development of more potent inhibitors of Ad37 binding and infection. The receptor for CVA24v, one of the main causative agents of AHC, has been unknown until now. We showed that this ocular virus, like Ad37, is also able to use SA as a receptor on corneal cells but not on conjunctival cells. This suggested that CVA24v may use two different receptors. As for Ad37, the receptor used by CVA24v on corneal cells also appears to be one or more sialic acid-containing glycoproteins. We believe that these findings may be a starting point for design and development of candidate drugs for topical treatment of AHC.

No description available.

Au cours de notre programme de thèse, nous avons isolé et caractérisé des molécules protéiques à activité antivirale intracellulaire dirigée contre le VIH-1. Ces protéines, appelées aRep, ont été obtenues par criblage d'une banque de protéines artificielles de nouvelle génération, construites de façon combinatoire à partir de protéines naturelles constituées de motifs alpha-hélicoidaux répétés. La cible virale, ou "appât", utilisé pour ce criblage a été une région de la polyprotéine Gag du VIH-1 identifiée comme une cible privilégiée de nouvelles thérapeutiques antivirales, car essentielle à l'assemblage viral, l'empaquetage du génome viral et le clivage de maturation de Gag aboutissant à la formation de virions infectieux. Deux molécules d'aRep à affinité élevée pour la cible virale, l'aRep4E3 (32 kDa; 7 motifs répétés) et l'aRep9A8 (28 kDa; 6 motifs répétés) ont ainsi été identifiées, isolées et clonées. L'étude de l'activité anti-VIH intracellulaire de ces aRep a été réalisée dans différents systèmes d'expression cellulaire, nécessitant la construction de lignées stables de cellules d'insecte et de cellules épithéliales humaines, et leur infection par différents types de vecteurs viraux recombinants, baculovirus ou lentivirus, porteurs du gène rapporteur luciférase. Mais surtout, cette étude a été menée sur des cellules lymphocytaires-T (SupT1), cibles naturelles du virus, exprimant ces aRep et infectées par du VIH-1 naturel infectieux. Nos résultats ont montré que l'aRep4E3 et l'aRep9A8 ont toutes deux un effet négatif significatif sur le cycle réplicatif du VIH-1, mais ciblent des fonctions virales différentes. L'aRep4E3 bloque l'empaquetage du génome viral, tandis que l'aRep9A8 inhibe la maturation et diminue l'infectivité virale. De plus, l'aRep9A8, exprimée de façon constitutive dans les cellules SupT1, leur confère une résistance au VIH: une lignée de SupT1 chroniquement infectée par le VIH-1 a pu être ainsi isolée et maintenue en culture pendant plusieurs semaines, sans effet cytopathique viro-induit apparent. Ces nouvelles données auront des implications non-négligeables dans le choix et la conduite de futures stratégies de thérapie cellulaire anti-VIH / HIV-1 infection is a long-term disease which required a long-life treatment. Besides the standard HAART regiment, HIV gene therapy is a promising alternative strategy which give rise to hope for the better HIV-1 treatment. Protein therapeutics is one another technique that represent high impact results in curing various types of disease. It is already become a significant part of current medical treatments. In this study we first designed aRep, a non-immunoglobulin scaffold protein which target two domains of HIV-1 Pr55Gag, SP1-NC and investigated their roles as an intracellular therapeutic agents. Phage display technology was used for the specific isolation of aRep against a critical C-terminal region of the HIV-1 Pr55Gag precursor from a large and diverse library. The antiviral activity of these two Pr55Gag binders was investigated using different cell systems. Two aRep scaffolds aRep4E3 and aRep9A8 were isolated and characterized. aRep4E3 contains 7 repeat motifs (32 kDa), meanwhile aRep9A8 has 6 repeat motifs (28 kDa). These two scaffold molecules found to be able to display antiviral effects on the late stage of HIV-1 replication, by reducing and delaying the viral progeny production. The difference in the molecular mechanism was observed between these two aRep proteins: aRep4E3 mainly interferes with the packaging of the viral genome, meanwhile aRep9A8 interferes with the proteolytic processing of Gag, and performs as a protease inhibitor to prevent the PR cleavage required for the production of newly infectious mature virus. Interestingly, aRep9A8 is able to survive in the chronical HIV-1 infected cells up to D38 pi with the low level of HIV-1 replication. Taken together, results suggested that aRep, a new type of scaffold protein could serve as a promising alternative agent in protein therapy, not only the HIV-1 infection but also the others pathogens or disorders

Sofosbuvir is a uridine based nucleotide inhibitor of the hepatitis C viral (HCV) polymerase that is the backbone of many treatment regimens. In combination with drugs targeting other viral enzymes (including the poorly potent guanosine analogue ribavirin or highly potent inhibitors of viral NS5A or protease) most patients clear virus and resistance to sofosbuvir is rare, allowing effective retreatment with sofosbuvir. Patients with Genotype 3 HCV respond less well than other genotypes and response is reduced in those previously exposed to interferon. Here we show that patientderived virus from patients with Genotype 3 HCV who relapse to sofosbuvir-based therapies have a reduced sensitivity to SOF in an in-vitro phenotyping assay. Analysis of viral sequencing data revealed two distinct polymorphisms (A150V and K206E) in the HCV polymerase that are associated with treatment failure and in-vitro; they reduce sofosbuvir sensitivity against genotype 3 hepatitis C virions. However both polymorphisms modify the cellular response to type I interferon and in cells lacking response to interferon the impact on sofosbuvir sensitivity is minimal. The A150V polymorphism reduces the response to interferon 70 fold whereas the K206E substitution has minimal effects on interferon in isolation but in combination with A150V reduces the response 100 fold. Preliminary data indicates that the A150V polymorphism interferes with the late response to type I interferons enabling the virus to overcome the induction of interferon-stimulated genes. These data indicate a complex interaction between direct acting antiviral drugs and the innate antiviral response.

Viruses are common causative agents of ocular infection among humans. Epidemic keratoconjuntivitis (EKC) is a severe and contagious ocular disease with reported outbreaks worldwide. It is estimated that this disease affects 20-40 million individuals every year, which leads to huge socioeconomic costs for the affected countries. EKC is characterized by keratitis and conjunctivitis but is also associated with pain, edema, lacrimation, and decreased vision that can prolong for months after the infection and in rare cases years. This disease is caused by human adenoviruses (HAdVs), which belong to the family of Adenoviridae. Currently, there is no available treatment against EKC. EKC is mainly caused by HAdV-8, HAdV-19, HAdV-37, HAdV-53, HAdV-54, and HAdV-56, which belong to species D HAdVs. HAdV-8, HAdV-19 and HAdV-37 have previously been shown to use sialic acid (SA)-containing glycans as cellular receptors to bind to and infect human corneal epithelial (HCE) cells. To characterize the receptor in more detail, we performed a glycan array, which included SA-containing glycans. A branched hexasaccharide terminating with SA in each arm was identified as a candidate receptor. This glycan corresponds to the glycan motif found on a ganglioside, GD1a. By performing a series of biological and biochemical experiments we confirmed the function of the GD1a glycan as a cellular receptor for EKC-causing HAdVs. However, the glycan used as a receptor was linked to plasma membrane protein(s) through O-glycosidic bonds, rather than to a lipid (as in the ganglioside). X-ray crystallography analysis showed that the two terminal SA:s interacted with two of the three previously identified SA-binding sites on the knob domain of the HAdV-37 capsid protein known as the fiber. Based on the structural features of the GD1a:HAdV-37 knob interaction, we assumed that a three-armed molecule with each arm terminating with SA would be an efficient inhibitor. Such molecules were designed, synthesized and found to efficiently prevent HAdV-37 binding to and infection of corneal cells. These results indicate that trisialic acids-containing compounds may be used for treatment of EKC. After binding to its primary receptor, most HAdVs have been shown to interact with αVβ3 and αVβ5 integrins to enter human cells. This interaction occurs through the RGD (arginine-alanine-aspartic acid) motif in the capsid protein known as the penton base. However, it was not clear if corneal epithelial cells express αVβ3 and αVβ5 integrins. Thus, to better understand additional early steps of infection by EKC-causing HAdVs, we performed binding and infection competition experiments using human corneal epithelial cells and siRNA, integrin specific antibodies, peptides and RGD-containing ligands indicating that α3, αV, β1 affected HAdV-37 infection of but not binding to HCE cells. We could also see that HAdV-37 co-localize with α3 and αV at after entry into HCE cells. In situ histochemistry confirmed that the expression of α3 and αV in human corneal tissue. Overall, our results suggest that αV and α3 integrins are important for HAdV-37 infection of corneal cells. Altogether, these results provide further insight into the biology of HAdVs and open up for development of novel antiviral drugs.

.La recurrencia de la infección por el VHC se produce de forma universal tras el trasplante hepático. La hepatopatía secundaria a la recurrencia de la infección tras el trasplante hepático evoluciona de forma más rápida que en sujetos inmunocompetentes y condiciona una peor supervivencia del injerto y del paciente.-El estudio de la cinética viral del VHC durante el trasplante y en la fase inmediatamente posterior. A través de este estudio se describió un descenso rápido de la carga viral en la fase anhepática y en la de reperfusión debido a la falta de producción de viriones y de aclaramiento hepático. Sin embargo, el ARN del VHC es detectable durante prácticamente todo el proceso y es a partir de estas partículas circulantes que se infecta el injerto. La replicación viral se inicia a las pocas horas tras la reperfusión hepática como lo demuestra el incremento de la carga viral que se produce en los días posteriores.-Eficacia y seguridad del tratamiento antiviral con interferón y ribavirina en pacientes cirróticos infectados por el VHC en lista de espera para trasplante hepático. Una de las estrategias propuestas para impedir la recidiva de la infección tras el TH es la erradicación del VHC en la fase pre-TH. La cirrosis avanzada constituye una contraindicación para el tratamiento antiviral con interferón y ribavirina. Sin embargo, con una selección adecuada de los pacientes y un seguimiento estrecho, la eficacia del tratamiento antiviral alcanzó un 30% (negativización del ARN-VHC).En un 20% de los pacientes del estudio se logró evitar la recidiva de la infección tras el trasplante-Recurrencia de la infección por el VHC tras el TH en receptores de órganos cadavéricos versus receptores de donante vivo. A través de este estudio prospectivo se demostró que la recurrencia de la infección por el VHC en receptores de donante vivo es significativamente más grave que en los receptores de órgano cadavérico. Las complicaciones biliares o el fenómeno de la regeneración hepática podrían justificar esta mayor agresividad. Estos resultados deberían tenerse en cuenta en la toma de decisiones por parte de los diferentes equipos de transplante ya que podría comprometer la supervivencia del injerto y del paciente. / ."Hepatitis C virus (HCV) infection recurrence after liver transplantation: prognostic factors for early and severe recurrence."HCV recurrence after liver transplantation (LT) is almost universal. HCV-related liver disease progresses more rapidly after liver transplantation than in immunocompetent individuals. Thus, survival after LT is also lower in these patients compared to other groups.-HCV virus kinetics during and immediately after LT. This study demonstrated a sharp decrease in viral load during the anhepatic phase and reperfusion, most likely owing to a lack of virions production and hepatic clearance.However, HCV-RNA is detectable during almost all the surgical procedure and circulating virions are supposed to cause graft infection.Viral replication begins immediately after graft reperfusion as demonstrated by the rapid increase in viral load during the first days after transplantation.-Efficacy and safety of antiviral therapy in HCV-cirrhotic patiens awaiting liver transplantation. One of the strategies that may avoid HCV-recurrence after LT is to erradicate viral infection before the surgery. Antiviral therapy is contraindicated in decompensated cirrhotic patients because its relative low efficacy and high risk of adverse events. However, we have demonstrated that an accurate selection and follow up of patients can lead to a succesful outcome in 30% of them (HCV-negativization during treatment). HCV-recurrence after LT was avoided in 20% of patients.-HCV-recurrence after living donor and cadaveric donor liver transplantation. In this prospective study we observed that HCV recurrence was significantly more severe in living donor liver transplantation compared to cadaveric liver transplantation.Type of donor (living vs cadaveric) was an independent prognostic factor for severe HCV-recurrence. Biliary complications and liver regeneration may play a role in this more severe outcome of HCV recurrence after living donor liver transplantation.These results should be taken into account in the decission-making process of transplant programs, since severe HCV-recurrence may ultimately compromise graft and patient survival.