Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.Chronic hepatitis C virus (HCV) infection affects millions of people worldwide and poses a major threat to human health (42). However, efforts to understand HCV pathogenesis and identify specific HCV antivirals to supplement or substitute for current interferon (IFN)-based therapies have been impeded by the lack of a robust, fully permissive tissue culture system and the absence of small, convenient animal models of HCV infection. While the former problem has been partially solved recently by the development of systems allowing productive HCV infection in human hepatoma cells (25,45,49,51), the chimpanzee remains the only well-validated animal model that is susceptible to HCV infection. Several drawbacks, however, including availability, ethical considerations, and extraordinary cost, limit use of the chimpanzee for modeling HCV infection and confirming the activity of candidate antivirals.

Immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed at the conserved inner core protein VP6 of rotavirus, such as the IgA7D9 MAb, provide protective immunity in adult and suckling mice when delivered systemically. While these antibodies do not have traditional in vitro neutralizing activity, they could mediate their antiviral activity either by interfering with the viral replication cycle along the IgA secretory pathway or by acting at mucosal surfaces as secretory IgA and excluding virus from target enterocytes. We sought to determine the critical step at which antirotaviral activity was initiated by the IgA7D9 MAb. The IgA7D9 MAb appeared to directly interact with purified triple-layer viral particles, as shown by immunoprecipitation and immunoblotting. However, protection was not conferred by passively feeding mice with the secretory IgA7D9 MAb. This indicates that the secretory IgA7D9 MAb does not confer protection by supplying immune exclusion activity in vivo. We next evaluated the capacity of polymeric IgA7D9 MAb to neutralize rotavirus intracellularly during transcytosis. We found that when polymeric IgA7D9 MAb was applied to the basolateral pole of polarized Caco-2 intestinal cells, it significantly reduced viral replication and prevented the loss of barrier function induced by apical exposure of the cell monolayer to rotavirus, supporting the conclusion that the antibody carries out its antiviral activity intracellularly. These findings identify a mechanism whereby the well-conserved immunodominant VP6 protein can function as a target for heterotypic antibodies and protective immunity.Rotavirus (RV) is the main cause of severe diarrhea in young infants worldwide and is responsible for 611,000 deaths per year (36). Two vaccines based on attenuated live viruses have recently completed successful phase III clinical trials and showed efficacy in preventing severe RV diarrhea caused by multiple serotypes (45, 50). However, the relevant immune effectors required for achieving protection against diverse circulating strains are still a matter of considerable debate. While CD8 ϩ T cells facilitate the timely clearance of rotavirus infection, B cells with the appropriate pattern of mucosal homing receptors and antibody production have been demonstrated as crucial for protection from reinfection (17, 24).Neutralizing antibodies against RV that block viral replication in cell culture are felt to play a major role in defense. A variety of passive antibody transfer studies in animals (28), as well as immunization studies using selected rotavirus reassortants in humans (7), have demonstrated a clear role for VP4 and VP7 in protective immunity. Neutralizing antibodies are directed against the outer shell VP4 and VP7 proteins, whose variable antigenic specificities define multiple serotypes, assigned as P and G serotypes, respectively (41). Worldwide, the G1-to-G4 serotypes associated with P4 and P8 and the more recently described G9/P6 or P8 serotypes are globally important rotavirus G/P combinations in human...

CC chemokine receptor 5 (CCR5) is a receptor for chemokines and the coreceptor for R5 HIV-1 entry into CD4(+) T lymphocytes. Chemokines exert anti-HIV-1 activity in vitro, both by displacing the viral envelope glycoprotein gp120 from binding to CCR5 and by promoting CCR5 endocytosis, suggesting that they play a protective role in HIV infection. However, we showed here that different CCR5 conformations at the cell surface are differentially engaged by chemokines and gp120, making chemokines weaker inhibitors of HIV infection than would be expected from their binding affinity constants for CCR5. These distinct CCR5 conformations rely on CCR5 coupling to nucleotide-free G proteins ((NF)G proteins). Whereas native CCR5 chemokines bind with subnanomolar affinity to (NF)G protein-coupled CCR5, gp120/HIV-1 does not discriminate between (NF)G protein-coupled and uncoupled CCR5. Interestingly, the antiviral activity of chemokines is G protein independent, suggesting that "low-chemokine affinity" (NF)G protein-uncoupled conformations of CCR5 represent a portal for viral entry. Furthermore, chemokines are weak inducers of CCR5 endocytosis, as is revealed by EC50 values for chemokine-mediated endocytosis reflecting their low-affinity constant value for (NF)G protein-uncoupled CCR5. Abolishing CCR5 interaction with (NF)G proteins eliminates high-affinity binding of CCR5 chemokines but preserves receptor endocytosis, indicating that chemokines preferentially endocytose low-affinity receptors. Finally, we evidenced that chemokine analogs achieve highly potent HIV-1 inhibition due to high-affinity interactions with internalizing and/or gp120-binding receptors. These data are consistent with HIV-1 evading chemokine inhibition by exploiting CCR5 conformational heterogeneity, shed light into the inhibitory mechanisms of anti-HIV-1 chemokine analogs, and provide insights for the development of unique anti-HIV molecules

CCR5 plays immune functions and is the coreceptor for R5 HIV-1 strains. It exists in diverse conformations and oligomerization states. We interrogated the significance of the CCR5 structural diversity on HIV-1 infection. We show that envelope glycoproteins (gp120s) from different HIV-1 strains exhibit divergent binding levels to CCR5 on cell lines and primary cells, but not to CD4 or the CD4i monoclonal antibody E51. This owed to differential binding of the gp120s to different CCR5 populations, which exist in varying quantities at the cell surface and are differentially expressed between different cell types. Some, but not all, of these populations are antigenically distinct conformations of the coreceptor. The different binding levels of gp120s also correspond to differences in their capacity to bind CCR5 dimers/oligomers. Mutating the CCR5 dimerization interface changed conformation of the CCR5 homodimers and modulated differentially the binding of distinct gp120s. Env-pseudotyped viruses also use particular CCR5 conformations for entry, which may differ between different viruses and represent a subset of those binding gp120s. In particular, even if gp120s can bind both CCR5 monomers and oligomers, impairment of CCR5 oligomerization improved viral entry, suggesting that HIV-1 prefers monomers for entry. From a functional standpoint, we illustrate that the nature of the CCR5 molecules to which gp120/HIV-1 binds shapes sensitivity to inhibition by CCR5 ligands and cellular tropism. Differences exist in the CCR5 populations between T-cells and macrophages, and this is associated with differential capacity to bind gp120s and to support viral entry. In macrophages, CCR5 structural plasticity is critical for entry of blood-derived R5 isolates, which, in contrast to prototypical M-tropic strains from brain tissues, cannot benefit from enhanced affinity for CD4. Collectively, our results support a role for CCR5 heterogeneity in diversifying the phenotypic properties of HIV-1 isolates and provide new clues for development of CCR5-targeting drugs.

No abstract

Chemokines promote the recruitment of leukocytes to sites of infection and inflammation by activating conventional heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). Chemokines are also recognized by a set of atypical chemokine receptors (ACRs), which cannot induce directional cell migration but are required for the generation of chemokine gradients in tissues. ACRs are presently considered "silent receptors" because no G protein-dependent signaling activity is observed after their engagement by cognate ligands. We report that engagement of the ACR D6 by its ligands activates a β-arrestin1-dependent, G protein-independent signaling pathway that results in the phosphorylation of the actin-binding protein cofilin through the Rac1-p21-activated kinase 1 (PAK1)-LIM kinase 1 (LIMK1) cascade. This signaling pathway is required for the increased abundance of D6 protein at the cell surface and for its chemokine-scavenging activity. We conclude that D6 is a signaling receptor that exerts its regulatory function on chemokine-mediated responses in inflammation and immunity through a distinct signaling pathway.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. In this study, we investigated RNA interference (RNAi), a specific gene silencing process mediated by small interfering RNA (siRNA) duplexes, as an antiviral strategy against HCV. Synthetic siRNAs were designed to target conserved sequences of the HCV 5' nontranslated region (NTR) located in a functional, stem-loop structured domain of the HCV internal ribosome entry site (IRES), which is crucial for initiation of polyprotein translation. Several siRNAs dramatically reduced or even abrogated the replication of selectable subgenomic HCV replicons upon cotransfection of human hepatoma cells with viral target and siRNAs, or upon transfection of cells supporting autonomous replication of HCV replicon with siRNAs. Importantly, three siRNAs also proved capable of strongly inhibiting virus production in cell culture. One siRNA, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. These results indicate that the HCV life cycle can be efficiently blocked by using properly-designed siRNAs that target functionally important, highly conserved sequences of the HCV IRES. This finding offers a novel approach towards developing IRES-based antiviral treatment for chronic HCV infections.

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.