Neutralizing Antibodies Targeting BK Polyomavirus: Clinical Importance and Therapeutic Potential for Kidney Transplant Recipients.

Most of the world's adult population is latently infected by the BK polyomavirus. It causes asymptomatic infection in healthy individuals but emerged as a threat to kidney transplant recipients because of virus-associated nephropathy caused by immunosuppressive therapy. In these conditions, when a functional cellular response is impaired by immunosuppression, neutralizing antibodies may play a major role because they can directly prevent infection of target cells, independently of cell-mediated immunity, by binding to the viral particles. Studying the contribution of anti-BK virus neutralizing antibodies in viral control has long been hampered by the lack of convenient in vitro models, but major progress has been made in the past decade. The four BK virus genotypes have been demonstrated to behave as distinct serotypes. A low recipient neutralizing antibody titer against the donor's serotype before kidney transplant has been significantly associated with BK virus replication after transplant. Different mechanisms exploited by the BK virus to evade neutralizing antibodies have been described. Recent studies also support the potential benefit of administering intravenous Igs or monoclonal neutralizing antibodies as a therapeutic strategy, and more interestingly, this could also be used as preventive or preemptive therapy before advanced kidney damage has occurred. Besides, neutralizing antibodies could be induced by vaccination. In this review, we summarize accumulated knowledge on anti-BK virus neutralizing antibodies as well as their clinical importance and therapeutic potential for kidney transplant recipients.

Molecular epidemiology and risk factors associated with BK and JC polyomavirus urinary shedding after kidney allograft.

Polyomaviruses BK (BKPyV) and JC (JCPyV), belonging to the Polyomaviridae, are responsible for human pathologies. In kidney transplant recipients, BKPyV replication can lead to irreversible nephron damage whereas JCPyV replication remains asymptomatic. Concomitant replication is rare and potential competition between the infections has been described. The aim of this retrospective case-control study was to describe the molecular epidemiology and risk factors associated with BKPyV and JCPyV replication in a cohort of kidney transplant recipients. In total, 655 urine samples from 460 patients were tested for BKPyV and JCPyV DNA. Positive samples were submitted to strain genotyping. Demographic and clinical characteristics were also compared. Isolated JCPyV and BKPyV was found in 16.5% and 23.3% of patients, respectively; co-replication was rare (3.9%). BKPyV strains Ib-2, Ib-1, and IVc-2 were the most prevalent. JCPyV strains mostly belonged to genotypes 4 and 1B. During follow-up, JCPyV shedding significantly reduced the risk of BKPyV DNAuria, with an odds ratio of 0.57 (95% confidence interval: 0.35-0.99), and was associated with better prognosis than BKPyV replication, based on the estimated glomerular filtration rate. Molecular epidemiology of BKPyV and JCPyV strains in our region was similar to previous studies. This study suggests that JCPyV is benign and appears to limit damaging BKPyV replication. JCPyV DNAuria screening could thus be a useful strategy to predict BKPyV-related outcomes.

BK Polyomavirus bkv-miR-B1-5p: A Stable Micro-RNA to Monitor Active Viral Replication after Kidney Transplantation.

BACKGROUND: Bkv-miR-B1-5p is a viral micro-RNA (miRNA) specifically produced during BK polyomavirus (BKPyV) replication. Recent studies have suggested using bkv-miR-B1-5p as a biomarker to monitor viral infection and predict complications in kidney transplant patients. To identify the technical limitations of this miRNA quantification in biological samples, knowledge of its stability and distribution in the extracellular compartment is necessary. Moreover, a proof of concept for using bkv-miR-B1-5p as a biomarker of active replication in chronic infection is still missing in the published literature. METHODS: The stability of bkv-miR-B1-5p was evaluated in samples derived from cell cultures and in urine from BKPyV-infected kidney transplant recipients. The miRNA was quantified in different fractions of the extracellular compartment, including exosomes, and protein binding was evaluated. Finally, we developed an in vitro model for chronic culture of BKPyV clinical isolates to observe changes in the bkv-miR-B1-5p level during persistent infections. RESULTS: Bkv-miR-B1-5p is a stable biomarker in samples from humans and in vitro experiments. Marginally associated with the exosomes, most of the circulating bkv-miR-B1-5p is bound to proteins, especially Ago2, so the miRNA quantification does not require specific exosome isolation. The bkv-miR-B1-5p level is predictable of viral infectivity, which makes it a potential specific biomarker of active BKPyV replication after kidney transplantation.

BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission.

Most people are asymptomatic carriers of the BK polyomavirus (BKPyV), but the mechanisms of persistence and immune evasion remain poorly understood. Furthermore, BKPyV is responsible for nephropathies in kidney transplant recipients. Unfortunately, the sole therapeutic option is to modulate immunosuppression, which increases the risk of transplant rejection. Using iodixanol density gradients, we observed that Vero and renal proximal tubular epithelial infected cells release two populations of infectious particles, one of which cosediments with extracellular vesicles (EVs). Electron microscopy confirmed that a single vesicle could traffic tens of viral particles. In contrast to naked virions, the EV-associated particles (eBKPyVs) were not able to agglutinate red blood cells and did not use cell surface sialylated glycans as an attachment factor, demonstrating that different entry pathways were involved for each type of infectious particle. However, we also observed that naked BKPyV and eBKPyV were equally sensitive to neutralization by the serum of a seropositive patient or commercially available polyvalent immunoglobulin preparations, which occurred at a postattachment step, after endocytosis. In conclusion, our work shows a new mechanism that likely plays a critical role during the primary infection and in the persistence, but also the reactivation, of BKPyV.IMPORTANCE Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases.

Functional Study of the C-Terminal Part of the Hepatitis C Virus E1 Ectodomain.

In the hepatitis C virus (HCV) envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acid [aa] 270 and aa 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the region from aa 314 to aa 342 (the aa314-342 region), which has been shown to contain two α helices (α2 and α3) by nuclear magnetic resonance studies. Of the 22 generated mutants, 20 were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI coreceptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix aa 315 to 324 (315-324) residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its coreceptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry.IMPORTANCE HCV is a major public health problem worldwide. The virion harbors two envelope proteins, E1 and E2, which are involved at different steps of the viral life cycle. Whereas E2 has been extensively characterized, the function of E1 remains poorly defined. We characterized here the function of the putative fusion peptide and the region containing α helices of the E1 ectodomain, which had been previously suggested to be important for virus entry. We could confirm the importance of these regions for the virus infectivity. Interestingly, we found several residues modulating the virus's dependence on several HCV receptors, thus highlighting the role of E1 in the interaction of the virus with cellular receptors. Whereas mutations in the putative fusion peptide affected HCV infectivity and morphogenesis, several mutations in the α2-helix region led to a loss of infectivity with no effect on assembly, indicating a role of this region in virus entry.

Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly.

Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug-resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size-exclusion chromatography, we obtained from HCV-seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2 ), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low-density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma-derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine-binding sites in KIV7 , KIV8 , and KIV10 were not required. CONCLUSIONS: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851-1864).

High association of T1858-G1896 precore mutations with impaired base pairing and high hepatitis B virus DNA levels in HBeAg-negative chronically infected patients.

The progression of liver disease in hepatitis B virus (HBV) infection is fostered by active virus replication. Mutations in the basal core promoter (BCP) and precore (PC) regions of the HBV genome are known to have an impact on viral replication. The aim of the present study was to assess the correlation of mutation profiles in the BCP and PC regions with the viral load in HBeAg-negative chronically infected patients. The HBV genotype, BCP/PC mutations, serum HBV DNA levels, and associated serological markers were analyzed in 92 HBeAg-negative chronically infected patients. Sequence analysis of the BCP and PC regions revealed variability of 19% and 24.1%, respectively. This variability was primarily associated with five critical positions (1753, 1762, 1764, 1896 and 1899). An elevated HBV viral load (>20,000 IU/ml) was classically correlated with F2-F4 liver fibrosis, elevated serum alanine aminotransferase levels, 1762/1764 and 1753 combination mutations, and surprisingly, with an 1858T-1896G double mutation that impairs base pairing at the base of the bulge in the ε encapsidation signal. An analysis of covariance confirmed the independent nature of the relationship between the 1858T-1896G double mutation and the HBV viral load. In conclusion, independently of conventional parameters, this study demonstrates that a high serum HBV DNA level was also associated with PC 1858-1896 mutations. These BCP/PC mutations may have important clinical implications as predictive factors for HBV DNA increase.

Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action.

UNLABELLED: Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface. IMPORTANCE: In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors.

New Insights into the Understanding of Hepatitis C Virus Entry and Cell-to-Cell Transmission by Using the Ionophore Monensin A.

UNLABELLED: In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation. IMPORTANCE: Although several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to hepatic cells are still unclear. In our study, we used monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission pathways are both pH-dependent processes. We generated monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of hypervariable region 1 (HVR1) of the E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH dependency of cell-free and cell-to-cell transmission.

Regulation of core expression during the hepatitis C virus life cycle.

Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed kinetics of core expression were also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated with its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein, during the assembly step of the viral life cycle.

Comparative Evaluation of Three Nucleic Acid-Based Assays for BK Virus Quantification.

With the growing importance of BK virus (BKV), effective and efficient screening for BKV replication in plasma and urine samples is very important for monitoring renal transplant and hematopoietic stem cell transplant recipients, who are at increased risk of BKV-associated diseases. However, recent assays proposed by many manufacturers have not been tested, and the available tests have not been standardized. The aim of the present study was to evaluate and compare the performances of three commercially available kits, R-gene, GeneProof, and RealStar, on plasma and urine specimens from patients infected with various genotypes and to determine the correlations with the results from a reference laboratory. A qualitatively excellent global agreement (96.8%) was obtained. RealStar PCR tended to give a higher sensitivity, especially for subtype Ib1 samples. Comparison of 30 plasma samples and 53 urine samples showed a good agreement between the three assays, with Spearman's Rho correlation coefficient values falling between 0.92 and 0.98 (P < 0.001). Moreover, a perfect correlation was obtained for comparison of the assay performances with the AcroMetrix BKV panel (P < 0.001 for all comparisons). According to Bland-Altman analysis, more than 95% (240/249 comparisons) of sample comparisons were situated in the range of the mean ± 2 standard deviations (SD). The greatest variability between assays was observed for 10.2% of subtype Ib2 samples, with differences of >1 log10 copies/ml. In conclusion, this study demonstrated the reliable and comparable performances of the R-gene, GeneProof, and RealStar real-time PCR systems for quantification of BKV in urine and plasma samples. All three real-time PCR assays are appropriate for screening of BKV replication in patients.

Identification of conserved residues in hepatitis C virus envelope glycoprotein E2 that modulate virus dependence on CD81 and SRB1 entry factors.

UNLABELLED: In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies. IMPORTANCE: Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.

Analysis of serine codon conservation reveals diverse phenotypic constraints on hepatitis C virus glycoprotein evolution.

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes.

Disulfide bonds in hepatitis C virus glycoprotein E1 control the assembly and entry functions of E2 glycoprotein.

Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.

The antimalarial ferroquine is an inhibitor of hepatitis C virus.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti-HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)-free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell-cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell-free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell-to-cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. CONCLUSION: FQ is a novel, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals.

Epidemiology and Dynamics of BK Polyomavirus Replication after Kidney Transplantation.

BACKGROUND/OBJECTIVES: In the absence of an effective antiviral treatment for BK polyomavirus (BKPyV), a better understanding of the epidemiology and time course of BKPyV replication after kidney transplantation is needed to limit the virus's impact on the graft outcome. METHODS: In a 7-year study, we screened more than 430 kidney transplant recipients and analyzed the time course and virological characteristics of BKPyV replication. RESULTS: Urinary viral replication was observed in 116 (27%) of the 430 patients, and 90 of the 116 (78%) had viral DNAemia. Thirty-eight patients (8.8%) were presumed to have nephropathy (DNAemia > 4 log10 copies/mL). Of the patients with BKPyV replication, 48%, 60%, 71%, and 80% were first found to be positive one, two, three, and four months post-transplantation. The initial viral load in the urine was below 7 log10 copies/mL in 100% of the patients with viral replication first detected before the first month, and this proportion was 57% when viral replication was first detected after the first month. When the BKPyV replication was first detected in a urine sample at month 3 or later, 81.5% of patients had concomitant BKPyV DNAemia. The predominant viral subtype was Ib2 (60%), and there was no apparent relationship between the subtype and the time course of BKPyV replication. CONCLUSIONS: Urinary BKPyV replication occurs early after renal transplantation and in most patients will increase to a level requiring therapeutic intervention. Close monitoring for BKPyV in the early post-transplantation period would enable the pre-emptive adjustment of the immunosuppression regimen.

The Conserved YPX3L Motif in the BK Polyomavirus VP1 Protein Is Important for Viral Particle Assembly but Not for Its Secretion into Extracellular Vesicles.

The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.

Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes.

A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.