Molecular analysis of the massive GSH transport mechanism mediated by the human Multidrug Resistant Protein 1/ABCC1.

The transporter Multidrug Resistance Protein 1 (MRP1, ABCC1) is implicated in multidrug resistant (MDR) phenotype of cancer cells. Glutathione (GSH) plays a key role in MRP1 transport activities. In addition, a ligand-stimulated GSH transport which triggers the death of cells overexpressing MRP1, by collateral sensitivity (CS), has been described. This CS could be a way to overcome the poor prognosis for patients suffering from a chemoresistant cancer. The molecular mechanism of such massive GSH transport and its connection to the other transport activities of MRP1 are unknown. In this context, we generated MRP1/MRP2 chimeras covering different regions, MRP2 being a close homolog that does not trigger CS. The one encompassing helices 16 and 17 led to the loss of CS and MDR phenotype without altering basal GSH transport. Within this region, the sole restoration of the original G1228 (D1236 in MRP2) close to the extracellular loop between the two helices fully rescued the CS (massive GSH efflux and cell death) but not the MDR phenotype. The flexibility of that loop and the binding of a CS agent like verapamil could favor a particular conformation for the massive transport of GSH, not related to other transport activities of MRP1.

Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication.

Hepatitis Delta virus (HDV) is a satellite of Hepatitis B virus with a single-stranded circular RNA genome. HDV RNA genome synthesis is carried out in infected cells by cellular RNA polymerases with the assistance of the small hepatitis delta antigen (S-HDAg). Here we show that S-HDAg binds the bromodomain (BRD) adjacent to zinc finger domain 2B (BAZ2B) protein, a regulatory subunit of BAZ2B-associated remodeling factor (BRF) ISWI chromatin remodeling complexes. shRNA-mediated silencing of BAZ2B or its inactivation with the BAZ2B BRD inhibitor GSK2801 impairs HDV replication in HDV-infected human hepatocytes. S-HDAg contains a short linear interacting motif (SLiM) KacXXR, similar to the one recognized by BAZ2B BRD in histone H3. We found that the integrity of the S-HDAg SLiM sequence is required for S-HDAg interaction with BAZ2B BRD and for HDV RNA replication. Our results suggest that S-HDAg uses a histone mimicry strategy to co-activate the RNA polymerase II-dependent synthesis of HDV RNA and sustain HDV replication.

[Hepatitis delta virus replication and the role of the small hepatitis delta protein S-HDAg]. / Réplication du génome du virus de l'hépatite delta : un rôle pour la petite protéine delta S-HDAg.

Hepatitis delta virus (HDV) is a mammalian defective virus. Its genome is a small single-stranded circular RNA of approximately 1,680 nucleotides. To spread, HDV relies on hepatitis B virus envelope proteins that are needed for viral particle assembly and egress. Severe clinical features of HBV-HDV infection include acute fulminant hepatitis and chronic liver fibrosis leading to cirrhosis and hepatocellular carcinoma. One uniqueness of HDV relies on its genome similarity to viroids, small plant infectious uncoated RNAs. Devoid of viral replicase activity, HDV has to use host DNA-dependant RNA Pol II to replicate its genomic RNA. Thus, one can ask how does this replication occur? We describe first here the major steps of the viral RNA transcription and replication and then we detail the role of the Small HD protein in these processes, especially with regard to the Pol II recruitment.

Hepatitis B and hepatitis D virus infections in the Central African Republic, twenty-five years after a fulminant hepatitis outbreak, indicate continuing spread in asymptomatic young adults.

Hepatitis delta virus (HDV) increases morbidity in Hepatitis B virus (HBV)-infected patients. In the mid-eighties, an outbreak of HDV fulminant hepatitis (FH) in the Central African Republic (CAR) killed 88% of patients hospitalized in Bangui. We evaluated infections with HBV and HDV among students and pregnant women, 25 years after the fulminant hepatitis (FH) outbreak to determine (i) the prevalence of HBV and HDV infection in this population, (ii) the clinical risk factors for HBV and/or HDV infections, and (iii) to characterize and compare the strains from the FH outbreak in the 1980s to the 2010 HBV-HDV strains. We performed a cross sectional study with historical comparison on FH-stored samples (n = 179) from 159 patients and dried blood-spots from volunteer students and pregnant women groups (n = 2172). We analyzed risk factors potentially associated with HBV and HDV. Previous HBV infection (presence of anti-HBc) occurred in 345/1290 students (26.7%) and 186/870 pregnant women (21.4%)(p = 0.005), including 110 students (8.8%) and 71 pregnant women (8.2%), who were also HBsAg-positive (p = 0.824). HDV infection occurred more frequently in pregnant women (n = 13; 18.8%) than students (n = 6; 5.4%) (p = 0.010). Infection in childhood was probably the main HBV risk factor. The risk factors for HDV infection were age (p = 0.040), transfusion (p = 0.039), and a tendency for tattooing (p = 0.055) and absence of condom use (p = 0.049). HBV-E and HDV-1 were highly prevalent during both the FH outbreak and the 2010 screening project. For historical samples, due to storage conditions and despite several attempts, we could only obtain partial HDV amplification representing 25% of the full-length genome. The HDV-1 mid-eighties FH-strains did not form a specific clade and were affiliated to two different HDV-1 African subgenotypes, one of which also includes the 2010 HDV-1 strains. In the Central African Republic, these findings indicate a high prevalence of previous and current HBV-E and HDV-1 infections both in the mid-eighties fulminant hepatitis outbreak and among asymptomatic young adults in 2010, and reinforce the need for universal HBV vaccination and the prevention of HDV transmission among HBsAg-positive patients through blood or sexual routes.

HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes.

Hepatitis D virus (HDV) super-infection of Hepatitis B virus (HBV)-infected patients is the most aggressive form of viral hepatitis. HDV infection is not susceptible to direct anti-HBV drugs, and only suboptimal antiviral responses are obtained with interferon (IFN)-alpha-based therapy. To get insights on HDV replication and interplay with HBV in physiologically relevant hepatocytes, differentiated HepaRG (dHepaRG) cells, previously infected or not with HBV, were infected with HDV, and viral markers were extensively analyzed. Innate and IFN responses to HDV were monitored by measuring pro-inflammatory and interferon-stimulated gene (ISG) expression. Both mono- and super-infected dHepaRG cells supported a strong HDV intracellular replication, which was accompanied by a strong secretion of infectious HDV virions only in the super-infection setting and despite the low number of co-infected cells. Upon HDV super-infection, HBV replication markers including HBeAg, total HBV-DNA and pregenomic RNA were significantly decreased, confirming the interference of HDV on HBV. Yet, no decrease of circular covalently closed HBV DNA (cccDNA) and HBsAg levels was evidenced. At the peak of HDV-RNA accumulation and onset of interference on HBV replication, a strong type-I IFN response was observed, with interferon stimulated genes, RSAD2 (Viperin) and IFI78 (MxA) being highly induced. We established a cellular model to characterize in more detail the direct interference of HBV and HDV, and the indirect interplay between the two viruses via innate immune responses. This model will be instrumental to assess molecular and immunological mechanisms of this viral interference.

The hepatitis D satellite virus of hepatitis B virus: half-opening a new era to control viral infection?

PURPOSE OF REVIEW: To highlight new concepts and therapeutic approaches concerning hepatitis D virus (HDV) infection. RECENT FINDINGS: Common receptor for hepatitis B virus (HBV) and HDV has been elucidated, deciphering of HDV replication is still in progress, preliminary results of phase II proof-of-concept clinical assays for entry inhibitors and cellular farnesyl transferase inhibitors are now available. SUMMARY: Hepatitis D infection remains a severe acute and chronic liver illness with the only currently approved therapy (Peg-αIFN) achieving disappointingly low rates of sustained viral response and clinical improvement. Both sodium taurocolate cotransporting polypeptide and heparan sulphate glypican 5 are important for viral adsorption. Preliminary results of 6 months treatment with a subcutaneous HBV PreS1-derived myristoyled peptide as an entry inhibitor indicates an encouraging short-term response with low side-effects. In addition, the short-term use of oral farnesyl transferase inhibitors induces a log10 reduction of viral RNA in almost all treated patients, but is associated with gastrointestinal upset and weight loss (especially using 200 mg/day). Encouraging results are being reported using intravenous phosphorothioate nucleic acid polymers both in terms of HBV surface antigens (HBsAg) and HDV-RNA decline; interestingly, in some patients with a strong HBsAg decline, the appearance of anti-hepatitis Bs antibodies might suggest clinical end-point improvement.

Impact on antiviral resistance of E119V, I222L and R292K substitutions in influenza A viruses bearing a group 2 neuraminidase (N2, N3, N6, N7 and N9).

OBJECTIVES: While subtype-specific substitutions linked to neuraminidase (NA) inhibitor resistance are well described in human N1 and N2 influenza NAs, little is known about other NA subtypes. The aim of this study was to determine whether the R292K and E119V ±â€ŠI222L substitutions could be associated with oseltamivir resistance in all group 2 NAs and had an impact on virus fitness. METHODS: Reassortant viruses with WT NA or variant N2, N3, N6, N7 or N9 NAs, bearing R292K or E119V ±â€ŠI222L substitutions, were produced by reverse genetics. The antiviral susceptibility, activity, Km of the NA, mutation stability and in vitro virus fitness in MDCK cells were determined. RESULTS: NA activities could be ranked as follows regardless of the substitution: N3 ≥ N6 > N2 ≥ N9 > N7. Using NA inhibitor resistance interpretation criteria used for human N1 or N2, the NA-R292K substitution conferred highly reduced inhibition by oseltamivir and the N6- or N9-R292K substitution conferred reduced inhibition by zanamivir and laninamivir. Viruses with the N3- or N6-E119V substitution showed normal inhibition by oseltamivir, while those with the N2-, N7- or N9-E119V substitution showed reduced inhibition by oseltamivir. Viruses with NA-E119V + I222L substitutions showed reduced inhibition (N3 and N6) or highly reduced inhibition (N2, N7 and N9) by oseltamivir. Viruses bearing the NA-R292K substitution had lower affinity and viruses bearing the NA-E119V substitution had higher affinity for the MUNANA substrate than viruses with corresponding WT NA. CONCLUSIONS: NA-R292K and E119V + I222L substitutions conferred reduced inhibition by oseltamivir for all group 2 NAs. Surveillance of NA inhibitor resistance for zoonotic and human influenza viruses and the development of novel antiviral agents with different targets should be continued.

Protein-peptide arrays for detection of specific anti-hepatitis D virus (HDV) genotype 1, 6, and 8 antibodies among HDV-infected patients by surface plasmon resonance imaging.

Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg-anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome-viral etiology-exploring array.

Blocking human enterovirus 71 replication by targeting viral 2A protease.

OBJECTIVES: Human enterovirus 71 (EV-71), a member of the Enterovirus genus, constitutes a major public health issue in the Asia-Pacific region, where it is associated with several severe neurological complications. There is currently no effective vaccine or antiviral against EV-71. The aim of this study was to determine whether the six amino acid peptide LVLQTM, which was previously shown to inhibit human rhinovirus (HRV) 2A protease (2A(pro)) activity in vitro and HRV replication in vivo in mice, could be of more general use against enteroviruses and more particularly against EV-71. METHODS: To investigate whether the LVLQTM peptide was a pseudosubstrate of EV-71 2A(pro), a recombinant luciferase containing the LVLQTM sequence was designed so that recognition of this sequence by 2A(pro) led to luciferase activation. Direct interaction between EV-71 2A(pro) and the LVLQTM peptide was further confirmed by isothermal titration calorimetry. We then tested the effects of the peptide on EV-71 2A(pro) cleavage activity and EV-71 replication in HeLa cells. RESULTS: We showed that the LVLQTM peptide behaved as an effective substrate analogue of EV-71 2A(pro), which binds into the active site of the protease with a dissociation rate constant of 9.6 µM. Moreover, LVLQTM significantly inhibited eIF4G cleavage activity of 2A(pro) as well as EV-71 replication in HeLa cells. CONCLUSIONS: This study demonstrates that the LVLQTM peptide that has previously been shown to inhibit HRV replication is also an effective inhibitor of EV-71 2A(pro) and therefore of EV-71 replication, opening new doors in the development of new antivirals against EV-71.

Effects of mutations on herpes simplex virus 1 thymidine kinase functionality: an in vitro assay based on detection of monophosphate forms of acyclovir and thymidine using HPLC/DAD.

Discrimination between the mutations responsible for drug resistance and those of UL23 TK gene polymorphism can be difficult. A non-isotopic method has been developed to assess TK functionality by measuring monophosphate forms of both acyclovir (ACV) and thymidine using HPLC/DAD. Phenotypes of TKs could thus be characterized as TK altered (P84L, A189V, L227F), TK deficient (G200S, L291P) or TK partial (R163H). A reliable link between HSV UL23 TK mutations and ACV resistance is necessary for developing a powerful genotyping tool to detect ACV resistance quickly in clinical samples.

Ex vivo and in vivo inhibition of human rhinovirus replication by a new pseudosubstrate of viral 2A protease.

Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.

Refined study of the interaction between HIV-1 p6 late domain and ALIX.

The interaction between the HIV-1 p6 late budding domain and ALIX, a class E vacuolar protein sorting factor, was explored by using the yeast two-hybrid approach. We refined the ALIX binding site of p6 as being the leucine triplet repeat sequence (Lxx)4 (LYPLTSLRSLFG). Intriguingly, the deletion of the C-terminal proline-rich region of ALIX prevented detectable binding to p6. In contrast, a four-amino acid deletion in the central hinge region of p6 increased its association with ALIX as shown by its ability to bind to ALIX lacking the proline rich domain. Finally, by using a random screening approach, the minimal ALIX391-510 fragment was found to specifically interact with this p6 deletion mutant. A parallel analysis of ALIX binding to the late domain p9 from EIAV revealed that p6 and p9, which exhibit distinct ALIX binding motives, likely bind differently to ALIX. Altogether, our data support a model where the C-terminal proline-rich domain of ALIX allows the access of its binding site to p6 by alleviating a conformational constraint resulting from the presence of the central p6 hinge.

Genotypic detection of acyclovir-resistant HSV-1: characterization of 67 ACV-sensitive and 14 ACV-resistant viruses.

Infections due to herpes simplex virus (HSV) resistant to acyclovir (ACV) represent an important clinical concern in immunocompromised patients. In order to switch promptly to an appropriate treatment, rapid viral susceptibility assays are required. We developed herein a genotyping analysis focusing on thymidine kinase gene (TK) mutations in order to detect acyclovir-resistant HSV in clinical specimens. A total of 85 HSV-1 positive specimens collected from 69 patients were analyzed. TK gene could be sequenced directly for 81 clinical specimens (95%) and 68 HSV-1 specimens could be characterized as sensitive or resistant by genotyping (84%). Genetic characterization of 67 susceptible HSV-1 specimens revealed 10 polymorphisms never previously described. Genetic characterization of 14 resistant HSV-1 revealed 12 HSV-1 with either TK gene additions/deletions (8 strains) or substitutions (4 strains) and 2 HSV-1 with no mutation in the TK gene. DNA polymerase gene was afterwards explored. With this rapid PCR-based assay, ACV-resistant HSV could be detected directly in clinical specimens within 24 h.

Resistance of herpes simplex virus type 1 to acyclovir: thymidine kinase gene mutagenesis study.

By site-directed mutagenesis, we investigate the role of six mutations of herpes simplex virus type 1 thymidine kinase (TK) gene in the acquisition of resistance to acyclovir (ACV). TK activity was not impaired by substitutions located at codons 17, 161 and 374 and these mutations were thus related to TK gene polymorphism. Mutations His105Pro, Leu364Pro and Asp162Ala lead to the loss of TK activity that could result in ACV-resistance.

Selection of single-chain antibodies that specifically interact with vesicular stomatitis virus (VSV) nucleocapsid and inhibit viral RNA synthesis.

The RNA genome of non-segmented negative-strand RNA viruses is completely covered by the nucleoprotein (N) forming a ribonucleoprotein complex, the nucleocapsid. The nucleocapsid functions as the template for viral RNA synthesis that is mediated by a viral RNA-dependent RNA polymerase. It is postulated that the selection of molecules that would specifically target the nucleocapsid and thus inhibit the viral polymerase activity could represent a common approach to block negative-strand RNA viruses. Two single-chain antibody fragments (scFv) that were selected using the phage display technology and interacted specifically with vesicular stomatitis virus (VSV) nucleocapsid were characterized. The two recombinant antibodies recognize a conformational epitope on the nucleocapsid and immunoprecipitate specifically nucleocapsids from infected cell extracts. Both antibodies have a strong inhibitory effect on VSV transcription activity in vitro. Thus, they represent starting molecules for future development of in vivo viral RNA synthesis inhibitors.

Inhibition of ubiquitination and stabilization of human ubiquitin E3 ligase PIRH2 by measles virus phosphoprotein.

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally co-immunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by co-immunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

The HIV-1 Nef protein enhances the affinity of reverse transcriptase for RNA in vitro.

Several viral proteins, including nucleocapsid protein, integrase, Vif, Tat, and Nef have been proposed to act as cofactors of HIV-1 reverse transcription. Using two viral RNA probes, one overlapping the primer-binding site (PBS) and the other representing the ribosomal frameshifting signal (FS) of HIV-1 RNA, we found that recombinant full-length Nef protein (NefLAI) increased the affinity of reverse transcriptase (RT) for RNA in vitro, and interacted directly with RT in protein co-precipitation assays. The effect on RT-RNA binding and the capacity of Nef to interact with RT was also observed with N-terminal deletion mutant NefDelta57 and NefSF2, although to a lesser level. NefDelta57 corresponded to the processed Nef protein present in the internal core of mature virions, and lacked the N-myristoylated N-terminus and N-terminal region implicated in virus infectivity and pathogenicity in vivo. NefSF2, a Nef allele from a highly pathogenic strain of HIV-1, differed from NefLAI by the amino acid sequence and immunoreactivity of its N-terminal domain. The effect observed with NefSF2 and NefDelta57, and data from phage biopanning experiments suggested that the RT-binding region in Nef involved the C-terminal flexible loop of its C-terminal domain, but the function in RT-RNA binding was also influenced by its N-terminal domain.