Alexio Capovilla

Putative role of hepatitis B virus X protein in hepatocarcinogenesis: effects on apoptosis, DNA repair, mitogen-activated protein kinase and JAK/STAT pathways.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV‐induced malignant transformation is, however, incompletely understood. HBx, the protein encoded by the X open reading frame, is a transcriptional activator that has been implicated in hepatocarcinogenesis. HBx inhibits the function of the tumour suppressor protein p53 in what is thought to be an early event in hepatocyte transformation before the later accumulation of inactivating p53 point mutations. HBx inhibits apoptosis but also exerts pro‐apoptotic effects. The effects of HBx on apoptosis may be important not only for the development of HCC but also for the establishment of HBV infection. Further implication of HBx in hepatocyte transformation has been the demonstration that it inhibits the repair of damaged hepatocyte DNA. This effect may be mediated by interaction with p53 or through binding to the damaged DNA binding protein (DDB), which plays an accessory role in nucleotide excision repair. In addition, HBx activates cell signalling cascades involving mitogen‐activated protein kinase (MAPK) and Janus family tyrosine kinases (JAK)/signal transducer and activators of transcription (STAT) pathways. The implications of these modulating effects of HBx are not fully understood, but they are likely to have wide‐ranging effects on hepatocyte proliferation, apoptosis and the regulation of cell growth checkpoints. The cellular functions ascribed to HBx are unusually diverse, and defining the biologically important role of HBx during HBV replication will go some way to understanding the sequelae of chronic HBV infection.

Emergence of X4 usage among HIV-1 subtype C: evidence for an evolving epidemic in South Africa.

This study investigated the genotype and phenotype of HIV-1 isolates of 20 South African AIDS patients. We found the highest percentage of CXCR4 usage among primary isolates, in which 30% efficiently utilized CXCR4 and exhibited the syncytium-inducing phenotype. Phylogenetic analysis of env confirmed that 19 of the 20 were subtype C, and syncytium-inducing viruses had genetic changes in the V3 loop, characteristic of CXCR4 usage. Results imply that the frequency of CXCR4-utilizing subtype C is increasing with time.

The Efficacy of Generating Three Independent Anti-HIV-1 siRNAs from a Single U6 RNA Pol III-Expressed Long Hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.

Genotypic characterization and comparison of full-length envelope glycoproteins from South African HIV type 1 subtype C primary isolates that utilize CCR5 and/or CXCR4.

CCR5 has preferentially been used by all circulating HIV-1 subtype C viruses for cell entry. Recently, we reported the highest proportion of CXCR4-utilizing primary isolates among a cohort of 20 South African AIDS patients. This study describes and compares the Env genotypic characteristics from these 20 HIV-1 subtype C (and unique CD recombinant) primary isolates. Fourteen primary isolates utilized CCR5, four (including the CD recombinant) used CXCR4, and two were dual tropic. Extensive analysis and comparison of important structural motifs such as the N-linked glycosylation sites, signal sequences, CD4-binding sites, variable loops, cleavage sites, known neutralizing antibody and small molecule inhibitor binding sites confirmed that other than the expected differences in the V3 loop, no sequence motifs distinguished between R5 and X4 tropism. Further correlation of the env genotype to functionally relevant motifs is necessary to elucidate the relationship between biologically and immunologically relevant sites and aid vaccine and novel drug design.

Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser60 on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys126–Cys196), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys60 and gp120 Cys126, and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.

Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization.

Background: Interaction between HIV gp120 and cell CD4 initiates viral infection of host cells. Results: Only CD4 with reduced disulfides in domain 1 or 2 binds gp120, which inhibits thioredoxin-dependent CD4 dimerization. Conclusion: Cell surface oxidoreductases may prime CD4 for gp120 engagement, and impairment of redox-driven CD4 dimerization by gp120 may compromise CD4 function. Significance: Redox-dependent isomerization of CD4 is critical for HIV entry. Human CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes, where it plays a key role in the activation of immunostimulatory T cells and acts as the primary receptor for human immunodeficiency virus (HIV) glycoprotein (gp120). Although growing evidence suggests that redox exchange reactions involving CD4 disulfides, potentially catalyzed by cell surface-secreted oxidoreductases such as thioredoxin (Trx) and protein disulfide isomerase, play an essential role in regulating the activity of CD4, their mechanism(s) and biological utility remain incompletely understood. To gain more insights in this regard, we generated a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, and used these to show that while protein disulfide isomerase has little capacity for 2dCD4 reduction, Trx reduces 2dCD4 highly efficiently, catalyzing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulfide-linked 2dCD4 dimer. Moreover, we show that HIV gp120 is incapable of binding a fully oxidized, monomeric 2dCD4 in which both domain 1 and 2 disulfides are intact, but binds robustly to reduced counterparts that are the ostensible products of Trx-mediated isomerization. Finally, we demonstrate that Trx-driven dimerization of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is impaired when CD4 is bound to gp120. These observations reinforce the importance of cell surface redox activity for HIV entry and posit the intriguing possibility that one of the many pathogenic effects of HIV may be related to gp120-mediated inhibition of oxidoreductive CD4 isomerization.

The rare TXNRD1_v3 ("v3") splice variant of human thioredoxin reductase 1 protein is targeted to membrane rafts by N-acylation and induces filopodia independently of its redox active site integrity.

Background: The TXNRD1_v3 (“v3”) protein is a rare variant of human thioredoxin reductase 1. Results: Membrane targeting of v3 occurs by N-terminal myristoylation and palmitoylation, and its overexpression triggers induction of filopodia independently of its redox active site integrity. Conclusion: The v3 protein is targeted to membrane rafts. Significance: These results imply that v3, shown to be targeted to membrane rafts, may be involved in signaling events. The human selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene, is a key player in redox regulation. Alternative splicing generates several TrxR1 variants, one of which is v3 that carries an atypical N-terminal glutaredoxin domain. When overexpressed, v3 associates with membranes and triggers formation of filopodia. Here we found that membrane targeting of v3 is mediated by myristoylation and palmitoylation of its N-terminal MGC motif, through which v3 specifically targets membrane rafts. This was suggested by its localization in cholera toxin subunit B-stained membrane areas and also shown using lipid fractionation experiments. Utilizing site-directed mutant variants, we also found that v3-mediated generation of filopodia is independent of the Cys residues in its redox active site, but dependent upon its membrane raft targeting. These results identify v3 as an intricately regulated protein that expands TXNRD1-derived protein functions to the membrane raft compartment.

A modeled structure of an aptamer-gp120 complex provides insight into the mechanism of HIV-1 neutralization.

The HIV-1 envelope glycoprotein, gp120, is a key target for a class of drugs called entry inhibitors. Here we used molecular modeling to construct a three-dimensional model of an anti-gp120 RNA aptamer, B40t77, alone and in complex with gp120. An initial model of B40t77 was built from the predicted secondary structure and then subjected to a combination of energy minimization and molecular dynamics. To model the B40t77-gp120 complex, we docked the B40t77 predicted structure onto the CD4-induced epitope of the gp120 crystal structure. A series of gp120 point mutations in the predicted B40t77-gp120 interface were measured for their binding affinity for B40t77 by surface plasmon resonance. According to the model, of the 10 gp120 amino acids that showed a reduction in the level of binding when mutated to alanine, all of them are modeled as making direct contact with B40t77 as part of a hydrogen bonding network. Comparison by electron microscopy of the B40t77-gp120 complex with gp120 alone revealed that only the longest dimension of the complex significantly increased in length, in a manner consistent with the predicted model. Binding assays revealed that B40t77 can weaken the binding of gp120 to the monoclonal antibodies B6, B12, and 2G12, none of which have binding sites that overlap with B40t77, as well as strengthen the binding to the antibody 19b. Thus, B40t77 may induce distant conformational changes in gp120 that disrupt its association with host cells and may suggest a mechanism for aptamer neutralization of HIV-1.

Thioredoxin (Trx1) regulates CD4 membrane domain localization and is required for efficient CD4-dependent HIV-1 entry.

BACKGROUND CD4 is a glycoprotein expressed on the surfaces of certain immune cells. On lymphocytes, an important function of CD4 is to co-engage Major Histocompatibility Complex (MHC) molecules with the T Cell Receptor (TCR), a process that is essential for antigen-specific activation of T cells. CD4 localizes dynamically into distinct membrane microdomains, an important feature of its immunoregulatory function that has also been shown to influence the efficiency of HIV replication. However, the mechanism by which CD4 localization is regulated and the biological significance of this is incompletely understood. METHODS In this study, we used confocal microscopy, density-gradient centrifugation and flow cytometry to analyze dynamic redox-dependent effects on CD4 membrane domain localization. RESULTS Blocking cell surface redox exchanges with both a membrane-impermeable sulfhydryl blocker (DTNB) and specific antibody inhibitors of Thioredoxin-1 (Trx1) induces translocation of CD4 into detergent-resistant membrane domains (DRM). In contrast, Trx1 inactivation does not change the localization of the chemokine receptor CCR5, suggesting that this effect is targeted. Moreover, DTNB treatment and Trx1 depletion coincide with strong inhibition of CD4-dependent HIV entry, but only moderate reductions in the infectivity of a CD4-independent HIV pseudovirion. CONCLUSIONS Changes in the extracellular redox environment, potentially mediated by allosteric consequences of functional disulfide bond oxidoreduction, may represent a signal for translocation of CD4 into DRM clusters, and this sequestration, another potential mechanism by which the anti-HIV effects of cell surface oxidoreductase inhibition are exerted. GENERAL SIGNIFICANCE Extracellular redox conditions may regulate CD4 function by potentiating changes in its membrane domain localization.

Nucleotide excision repair by extracts of human fetal hepatocytes

Human hepatocytes are particularly exposed to genotoxins, and nucleotide excision repair (NER) in these cells is essential for the maintenance of genome integrity. To characterize NER under conditions that closely resemble the pathway in vivo, we report the preparation and use of primary human fetal liver extracts to define the repair of a 1,3‐intrastrand d(GpTpG)‐cisplatin DNA lesion. Endonucleolytic cleavage at unique sites on either side of the adduct occurs at similar positions to the dominant NER incisions that have been reported for HeLa extracts. However, incisions effected by primary hepatocyte extracts are more precise as no secondary cleavage sites are detected 5′ and 3′ to the cisplatin lesion.