Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach.     

Inhibition by α-Amanitin of Adenovirus 12 Replication in Human Embryo Kidney Cells and of Adenovirus Transformation of Hamster Cells

ADENOVIRUS infection of human embryonic kidney (HEK) cultures seems to induce cellular RNA synthesis, which is preceded by a transient increase in the activities of the Mg²⁺-activated and Mn²⁺-(NH₄)₂SO₄-activated DNA dependent RNA polymerases and in the rate of histone acetylation¹. The two polymerase reactions, assayed in isolated cell nuclei, apparently reflect the activities of distinct nucleolar and nucleo-plasmic RNA polymerases^2,3. We were therefore prompted to test the effect of a specific inhibitor of the mammalian DNA-dependent RNA polymerase function, α-amanitin, on the multiplication of adenovirus. α-Amanitin is a bicyclic octapeptide isolated from the poisonous mushroom Amanita phalloides⁴ and which blocks RNA synthesis in intact animals^5,6. Nuclei isolated from the livers of such animals show a reduced activity of the RNA polymerases associated with nucleoplasm^5,6 and the nucleolus⁶.     

Functional assessment of genetic variants of α1-Antitrypsin

Summary The specific activity of thirteen genetic variants of the protease inhibitor ₁-antitrypsin (₁-AT) has been determined. Elastase inhibitor activity was assayed using protein substrates (elastin and gelatin) and the synthetic substrate N-tert-butoxycarbonyl-l-alanine p-nitrophenyl ester. The synthetic substrate -N-benzoyl-dl-arginine p-nitroanilide HCl was used to assay trypsin inhibitor activity. The specific activity of ₁-AT was expressed as serum inhibition/immunological concentration of ₁-AT. Sera of PI type FM had reduced specific activity with elastase, but not with trypsin. With the possible exception of MP, no other variants showed significant differences in specific activity when compared with normal PI type M.This research was supported by the Medical Research Council of Canada (No. MA 5426)     

Three new rare variants of α1-Antitrypsin

Summary Three new rare genetic variants of the serum protein ₁-antitrypsin (₁-protease inhibitor) have been identified in a Caucasian population. The new alleles in the PI system are PI ^*EFRA, PT^*PCAS, and PI ^*XALB. When compared with the normal type M by isoelectric focusing in polyacrylamide, Efranklin (EFRA) is anodal, and Pcastoria (PCAS) and Xalban (XALB) are cathodal. These variants have been compared with previously described variants by isoelectric focusing and by electrophoresis in agarose and acid starch gels. All three variant alleles appear to be associated with normal amounts of ₁-antitrypsin, assayed both by functional and immunological methods.This work was supported by a grant from the Medical Research Council of Canada     

Inhibition Kinetics and the Aggregation of α-Glucosidase by Different Denaturants

Kinetic changes of alpha-glucosidase from Saccharomyces cerevisiae in guanidinium chloride (GdmCl) and SDS solutions were investigated. The results showed both denaturants can lead conformational changes and loss of enzymatic activities. However, the concentrations of denaturants causing loss of activities were much lower than that of conformational changes, which suggested that the conformation of active site of α-glucosidase was more fragile than the whole molecular conformation in response to the two denaturants. According to the different kinetic process of the enzyme in the GdmCl and SDS solutions, the further investigation on the process of denaturation were made, it showed GdmCl and SDS had different types of inhibition and different types of interaction with the enzyme. Furthermore, the mechanisms of the two denaturants were discussed.     

Inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds

Aggregation of alpha-synuclein (αS) into oligomers is critically involved in the pathogenesis of Parkinson's disease (PD). Using confocal single-molecule fluorescence spectroscopy, we have studied the effects of 14 naturally-occurring polyphenolic compounds and black tea extract on αS oligomer formation. We found that a selected group of polyphenols exhibited potent dose-dependent inhibitory activity on αS aggregation. Moreover, they were also capable of robustly disaggregating pre-formed αS oligomers. Based upon structure-activity analysis, we propose that the key molecular scaffold most effective in inhibiting and destabilizing self-assembly by αS requires: (i) aromatic elements for binding to the αS monomer/oligomer and (ii) vicinal hydroxyl groups present on a single phenyl ring. These findings may guide the design of novel therapeutic drugs in PD.     

Multifaceted Mechanisms of HIV-1 Entry Inhibition by Human α-Defensin

The human neutrophil peptide 1 (HNP-1) is known to block the human immunodeficiency virus type 1 (HIV-1) infection, but the mechanism of inhibition is poorly understood. We examined the effect of HNP-1 on HIV-1 entry and fusion and found that, surprisingly, this α-defensin inhibited multiple steps of virus entry, including: (i) Env binding to CD4 and coreceptors; (ii) refolding of Env into the final 6-helix bundle structure; and (iii) productive HIV-1 uptake but not internalization of endocytic markers. Despite its lectin-like properties, HNP-1 could bind to Env, CD4, and other host proteins in a glycan- and serum-independent manner, whereas the fusion inhibitory activity was greatly attenuated in the presence of human or bovine serum. This demonstrates that binding of α-defensin to molecules involved in HIV-1 fusion is necessary but not sufficient for blocking the virus entry. We therefore propose that oligomeric forms of defensin, which may be disrupted by serum, contribute to the anti-HIV-1 activity perhaps through cross-linking virus and/or host glycoproteins. This notion is supported by the ability of HNP-1 to reduce the mobile fraction of CD4 and coreceptors in the plasma membrane and to precipitate a core subdomain of Env in solution. The ability of HNP-1 to block HIV-1 uptake without interfering with constitutive endocytosis suggests a novel mechanism for broad activity against this and other viruses that enter cells through endocytic pathways.     

The Inhibition of α-Amylase by Tea Polyphenols

The inhibition of α-amylase from human saliva by polyphenolic components of tea and its specificity was investigated in vitro. Four kinds of green tea catechins, and their isomers and four kinds of their dimeric compounds (theaflavins) produced oxidatively during black tea production were isolated. They were (?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECg), (?)-epigallocatechin gallate (EGCg), (?)-catechin (C), (?)-gallocatechin (GC), (?)-catechin gallate (Cg), (?)-gallocatechin gallate (GCg), theaflavin (TF1), theaflavin monogallates (TF2A and TF2B), and theaflavin digallate (TF3). Among the samples tested, EC, EGC, and their isomers did not have significant effects on the activity of α-amylase. All the other samples were potent inhibitors and the inhibitory effects were in the order of TF3>TF2A>TF2B>TFl>Cg> GCg > ECg > EGCg. The inhibitory patterns were noncompetitive except for TF3.     

Inhibition of Hepatitis B Virus Replication by Rheum palmatum L. Ethanol Extract in a Stable HBV-producing Cell Line

Hepatitis B virus(HBV) infection is a severe health problem in the world. However, there is still not a satisfactory therapeutic strategy for the HBV infection. To search for new anti-HBV agents with higher efficacy and less side-effects, the inhibitory activities of traditional Chinese medicine Rheum palmatum L. ethanol extract(RPE) against HBV replication were investigated in this study. Quantitative real-time polymerase chain reaction(PCR) was employed to analyze the inhibitory activity of RPE against HBV-DNA replication in a stable HBV-producing cell line HepAD38; the expression levels of HBV surface antigen(HBsAg) and e antigen(HBeAg) were also determined by enzyme linked immunosorbent assay(ELISA) after RPE treatment. RPE could dose-dependently inhibit the production of HBV-DNA and HBsAg. The concentration of 50% inhibition(IC50) was calculated at 209.63, 252.53μg/mL, respectively. However, its inhibitory activity against HBeAg expression was slight even at high concentrations. RPE had a weak cytotoxic effect on HepAD38 cells(CC50 = 1 640μg/mL) and the selectivity index(SI) was calculated at 7.82. Compared with two anthraquinone derivatives emodin and rhein, RPE showed higher ability of anti-HBV and weaker cytotoxicity. So Rheum palmatum L. might possess other functional agents which could effectively inhibit HBV-DNA replication and HBsAg expression. Further purification of the active agents, identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.     

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase α-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).BK virus (BKV) is a human polyomavirus that was first isolated in the 1970s (15). Up to 90% of adults have serologic evidence of exposure to BKV, but in most humans the virus remains latent (25, 26). Almost all disease accompanied by BKV reactivation has been found in immunocompromised patients (22). In recent years, BKV has been associated with nephropathy (polyomavirus-associated nephropathy, or PVAN) in up to 10% of renal transplant patients. Once established, the disease results in allograft loss in 45 to 70% of the patients (18). Importantly, BKV preferentially replicates in human cells and less well in cells of other primates, and the virus is highly tumorigenic in rodents (21, 41, 44). This fact and the lack of sustainable viral replication in rodents or other convenient, experimental animal models have been an enormous setback to the study of PVAN.As with other members of the Polyomaviridae family, BKV virions are nonenveloped icosahedral particles with a diameter of 45 nm that contain a circular double-stranded DNA genome of 5.3 kb (1). In BKV and in other polyomaviruses, three genomic areas have been distinguished: (i) a noncoding control region including the origin of viral DNA replication, (ii) the early genes encoding large and small T antigens (TAgs), and (iii) the late genes which code for the capsid proteins VP-1, VP-2, and VP-3 and the agnoprotein (22).BKV DNA replication is similar to that of all other members of the Polyomaviridae family and requires only one viral protein, the multifunctional large TAg, whereas all other replication factors are supplied by the host (13, 14, 28, 39, 47). As the first step, TAg binds to the core origin, which contains the early palindrome, an AT-rich sequence, and the TAg binding site II, which consists of two pairs of G(G/A)GGC pentanucleotides. In the presence of ATP, TAg forms a double hexamer and partially melts the early palindrome (EP) and untwists the AT-rich sequence of the BKV core origin (5, 6, 14). Then the TAg double hexamers bidirectionally unwind the viral replication origin, which requires ATP hydrolysis. In the following process the two hexamers remain associated with each other, with the separated single-stranded DNA (ssDNA) threading through the hexameric channels (14). The viral core origin is sufficient to constitute a functional replication origin, but the presence of auxiliary domains increases its activity 5- to 100-fold in vivo (16, 30). After the viral TAg unwinds the core origin and its flanking sequences, replication protein A (RPA), the main eukaryotic ssDNA-binding protein, covers the resulting stretches of ssDNA, whereas topoisomerase I releases the resulting torsional stress and enhances initiation of DNA replication (5, 7, 43). Then, DNA polymerase α-primase (Pol-prim) is loaded onto this TAg-RPA-topoisomerase 1-DNA complex, yielding a functional initiation complex. In the following step, Pol-prim synthesizes short RNA primers at the origin, and these RNA primers are elongated by the DNA polymerase function of the enzyme complex (9, 35, 47). After a polymerase switch from Pol-prim to DNA polymerase δ (Pol δ) with the help of RPA, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA), processive DNA synthesis is completed by Pol δ in association with PCNA, the sliding clamp, on the leading strand (38, 51, 54, 59). Lagging-strand synthesis is discontinuous, and multiple initiation events catalyzed by Pol-prim must take place. Again, after the elongation of the RNA primers by Pol-prim, DNA synthesis is switched to Pol δ, which then synthesizes the complete Okazaki fragments. The maturation of these Okazaki fragments requires the collaboration of RNase H, PCNA, flap endonuclease 1 (Fen-1), Pol δ, and DNA ligase I to establish a continuous strand also on the lagging strand (9, 19, 20, 51, 55).TAg functions in infected cells rely heavily on specific associations with host proteins; for example, TAg interacts with RPA, Pol-prim, and topoisomerase I to replicate viral DNA. Selective interactions with the host p180 and p48 subunits of Pol-prim were shown to be responsible for species-specific replication of simian virus 40 (SV40) and murine polyomavirus (mPyV) DNAs, respectively (8, 47, 50). The subunits of Pol-prim are highly conserved since 88, 80, 89, and 90% of the amino acids are identical between human and murine p180, p68, p58, and p48, respectively. Biochemical studies have shown that TAg interacts independently with all four subunits of Pol-prim (8, 12, 57). Moreover, the p180, p58, and p48 subunits of Pol-prim also physically bind to RPA (7, 11, 57). RPA and TAg binding sites in the Pol-prim complex are essential for SV40 DNA replication in vitro since the presence of an excess of these purified binding peptides diminishes viral DNA replication in vitro (52, 53). Interestingly, species specificity requires the viral origin of DNA replication, whereas physical protein-protein interactions of purified protein complexes are not host specific in the absence of viral origin DNA (29, 42).Consistent with other polyomaviruses, analyses of BKV TAg-dependent DNA replication recently revealed that BKV DNA cannot be replicated in murine cells and that cell extracts are able to mimic this behavior (28). Furthermore, a BKV DNA replication system with the purified human proteins Pol-prim, RPA, topoisomerase I, and BKV TAg was inhibited by murine extracts, whereas SV40 DNA replication was not. Further investigations revealed that the presence of inhibitory activities (IAs) in extracts from murine cells blocks BKV DNA replication at an early step of TAg-mediated unwinding of the BKV origin of replication. Detailed analyses using the BKV monopolymerase DNA replication system, which we report here, show that Pol-prim functions as a species-specific factor associated with core origin functions. In addition, we reveal that the inhibitory activities in murine extracts, which are associated with origin-flanking sequence-dependent factor(s), regulate BKV DNA replication in murine cell extracts in a Pol-prim-independent manner.     

Proteolytic and N-Glycan Processing of Human α1-Antitrypsin Expressed in Nicotiana benthamiana

Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along the secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued the recombinant expression of the human protease inhibitor α1-antitrypsin (A1AT) in Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels of recombinant A1AT were expressed in leaves (up to 6 mg g⁻¹ of leaf material) in two forms: full-length A1AT located in the endoplasmic reticulum displaying inhibitory activity, and secreted A1AT processed in the reactive center loop, thus rendering it unable to interact with target proteinases. We found that the terminal protein processing is most likely a consequence of the intrinsic function of A1AT (i.e. its interaction with proteases [most likely serine proteases] along the secretory pathway). Secreted A1AT carried vacuolar-type paucimannosidic N-glycans generated by the activity of hexosaminidases located in the apoplast/plasma membrane. Notwithstanding, an intensive glycoengineering approach led to secreted A1AT carrying sialylated N-glycan structures largely resembling its serum-derived counterpart. In summary, we elucidate unique insights in plant glycosylation processes and show important aspects of postendoplasmic reticulum protein processing in plants.Recombinant protein-based drugs are among the fastest growing areas of development in the pharmaceutical industry. Consequently, there is a demand for exploring new production systems. Plants are increasingly being used for the expression of recombinant proteins, primarily because of their remarkable production speed and yield (for review, see Gleba et al., 2014). The highly conserved secretory pathway between human and plant cells allows similar, if not identical, protein folding, assembly, and posttranslational modifications. Importantly, plants are able to synthesize complex N-glycans, a prerequisite for the in vivo activity of many therapeutically interesting proteins. Despite substantial differences in N-glycan diversity, we and others have shown that plants are highly amendable to glycan engineering and allow proteins with controlled human-type N-glycosylation profiles to be generated (Castilho and Steinkellner, 2012). Moreover, it has even been possible to reconstruct entire human glycosylation pathways, which was shown by the introduction of the human sialylation and O-glycosylation processes in Nicotiana benthamiana (Castilho et al., 2010, 2012). These accomplishments render plants suitable for the production of human proteins that require a complex glycosylation profile.Notwithstanding, to use plants as a versatile expression host for complex human proteins, it is important to fully understand intracellular processes. Particularly detailed knowledge about constraints along the plant cell secretory pathway, including proteolytic processing, is required, because these constraints may compromise protein integrity and quality. Despite major achievements in controlling protein-bound oligosaccharide formation, some plant glycosylation peculiarities are not entirely understood. For example, plant cells synthesize so-called paucimannosidic N-glycans, a type of truncated glycans usually absent in mammals (Lerouge et al., 1998). The biosynthesis and physiological significance of this N-glycan formation has yet to be completely explained (Strasser et al., 2007; Liebminger et al., 2011). Another process not fully understood in plants is subcellular localization of proteins. Aberrant intracellular deposition and as a consequence, incorrect glycosylation of recombinant proteins are often reported. For example, recombinant proteins designed for secretion are frequently also located in the endoplasmic reticulum (ER) and as a consequence, carry oligomannosidic carbohydrates instead of the desired complex-type glycans (Loos et al., 2011; Schneider et al., 2014a). By contrast, KDEL-tagged proteins designed for ER retention are sometimes partially secreted (Van Droogenbroeck et al., 2007; Niemer et al., 2014). How and at which biosynthetic stage these plant-specific peculiarities arise are largely unpredictable, which makes controlled expression of recombinant proteins with features authentically to their natural counterparts a difficult task.One human protein that is pharmaceutically interesting, and thus needed in large amounts at high quality, is α1-antitrypsin (A1AT). This highly glycosylated protease inhibitor from the serpin superfamily interacts with a wide variety of proteases (Gettins, 2002). Like other serpins, A1AT is characterized by an exposed and mobile reactive center loop (RCL) with a Met (³⁵⁸M) residue acting as bait for specific target proteinases (Travis and Salvesen, 1983). The main biological role of plasma A1AT is to prevent excessive action of leukocyte-derived Ser proteinases, especially neutrophil elastase, in the circulatory system (Blank and Brantly, 1994). Therapeutic A1AT used in augmentation therapies is currently purified from pooled human serum, and the treatment can cost up to $100,000 per year per patient (Alkins and O’Malley, 2000). Concerns over the supply and safety of the products have urged searches for alternative recombinant sources of A1AT. Recombinant A1AT has been produced in human and nonhuman cell production systems with limited success (Blanchard et al., 2011; Brinkman et al., 2012; Ross et al., 2012; Lee et al., 2013). The production suffers from two major drawbacks: low expression levels and/or incorrect glycosylation (Garver et al., 1987; Chang et al., 2003; McDonald et al., 2005; Hasannia et al., 2006; Karnaukhova et al., 2006; Plesha et al., 2007; Agarwal et al., 2008; Nadai et al., 2009; Huang et al., 2010; Arjmand et al., 2011; Jha et al., 2012). The mature plasma-derived 52-kD protein has three N-linked glycosylation sites that are mainly decorated with disialylated structures (Kolarich et al., 2006). Sialylated N-glycans are a well-known requisite for the plasma half-life of A1AT (Mast et al., 1991; Lindhout et al., 2011; Lusch et al., 2013); the difficulties associated with obtaining them hamper the generation of biologically active A1AT in many expression systems.Here, we pursued the expression of recombinant human A1AT in glycoengineered N. benthamiana and investigated the system’s ability to generate active sialylated variants. Unusually high amounts of A1AT were obtained using a plant viral-based transient expression system. The inhibitor was efficiently secreted to the intercellular space (IF); however, peptide mapping showed that the secreted A1AT was truncated at both the N and C termini. Mass spectrometry (MS) -based N-glycan analysis of IF-derived A1AT showed that vacuolar typical paucimannosidic N-glycans were present. By expressing A1AT in Arabidopsis (Arabidopsis thaliana) knockout plants lacking β-N-acetylhexosaminidase (HEXO) activity (Liebminger et al., 2011), we showed that paucimannosidic structures are generated by the action of HEXO3 located at the plasma membrane.Coexpression with the mammalian genes necessary for in planta sialylation allowed the synthesis of disialylated A1AT, and sialylation levels could be increased by the synthesis of multiantennary glycans. By contrast, full-length A1AT purified from total soluble extracts exhibited ER-typical oligomannosidic carbohydrates. Using live-cell imaging, a GFP-tagged A1AT fusion did, indeed, exhibit aberrant ER-associated deposition of full-length A1AT. Elastase inhibition assays showed that ER-retained A1AT exhibits inhibitory activity, whereas the IF-derived truncated form was rendered inactive by cleavage within its RCL.     

Population genetics of α1-Antitrypsin in the Netherlands

Summary Two groups of 708 healthy blood donors and 563 patients affected with chronic obstructive lung disease (C.O.L.D.) respectively, have been screened for ₁-antitrypsin (₁AT) variants by electrophoresis on agarose-polyacrylamide gels at pH 4.7 and isoelectric focusing (IEF).The frequencies of the Pi (Protease inhibitor) alleles are comparable to those observed in the North European populations. As expected, the frequency of the Z gene is higher in the group of patients with C.O.L.D. Also the frequency of MZ phenotypes is higher among these patients, but in this case the difference is not statistically significant.With the aid of the electrophoretic methods described in the text we were able to detect a new electrophoretic variant (M₃) showing a mobility intermediate between the M₁ and the M₂ phenotypes.     

Aminoterminal Amphipathic α-Helix AH1 of Hepatitis C Virus Nonstructural Protein 4B Possesses a Dual Role in RNA Replication and Virus Production

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.     

Evaluation of Canonical siRNA and Dicer Substrate RNA for Inhibition of Hepatitis C Virus Genome Replication – A Comparative Study

Hepatitis C virus (HCV) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. The objective of this study was to test the ability of five Dicer substrate siRNAs (DsiRNA) to inhibit HCV replication and to compare these molecules to canonical 21 nt siRNA. DsiRNA molecules were designed to target five distinct regions of the HCV genome – the 5’ UTR and the coding regions for NS3, NS4B, NS5A or NS5B. These molecules were transfected into Huh7.5 cells that stably harboured an HCV subgenomic replicon expressing a firefly luciferase/neoR reporter (SGR-Feo-JFH-1) and were also tested on HCVcc-infected cells. All of the DsiRNAs inhibited HCV replication in both the subgenomic system and HCVcc-infected cells. When DsiRNAs were transfected prior to infection with HCVcc, the inhibition levels reached 99.5%. When directly compared, canonical siRNA and DsiRNA exhibited similar potency of virus inhibition. Furthermore, both types of molecules exhibited similar dynamics of inhibition and frequencies of resistant mutants after 21 days of treatment. Thus, DsiRNA molecules are as potent as 21 nt siRNAs for the inhibition of HCV replication and may provide future approaches for HCV therapy if the emergence of resistant mutants can be addressed.     

Inhibition of hepatic lipogenesis by α-cyano-4-hydroxycinnamate

In agreement with its well-known inhibition of mitochondrial carrier-mediated pyruvate transport, α-cyano-4-hydroxycinnamate elevates pyruvate and lactate levels in suspensions of isolated rat hepatocytes, whereas it lowers citrate levels and causes strongly depressed rates of fatty acid synthesis with glucose as carbon precursor. It stimulates the oxidation of exogenous fatty acids and inhibits their esterification.α-Cyano-4-hydroxycinnamate also impairs fatty acid synthesis from substrates (acetate, octanoate) that bypass mitochondrial pyruvate transport. Cholesterol synthesis from acetate, a process utilizing the same cytosolic acetyl-CoA pool as does fatty acid synthesis, is hardly affected by α-cyano-4-hydroxy-cinnamate. These observations suggest an inhibitory site of action of α-cyano-4-hydroxycinnamate located in the fatty-acid biosynthetic pathway itself. This suggestion has been confirmed by demonstrating the inhibition of purified rat-liver acetyl-CoA carboxylase by α-cyano-4-hydroxycinnamate at concentrations prevailing in the intact cell upon incubation with this compound.Maximal inhibition of purified acetyl-CoA carboxylase requires about 20 min of preincubation of the enzyme with α-cyano-4-hydroxycinnamate. Fatty acid synthesis from acetate in the intact cells is further diminished after an incubation time of 20 min.The inhibition by α-cyano-4-hydroxycinnamate of fatty acid synthesis from acetate can be partially overcome by insulin. Possible interactions of the inhibitor and the hormone at the level of acetyl-CoA carboxylase are discussed.It is concluded that α-cyano-4-hydroxycinnamate does not provide a simple and unequivocal tool to distinguish between actions of effectors on hepatic fatty acid synthesis per se and on the glycolytic pathway.     

Infection with “Escaped” Virus Variants Impairs Control of Simian Immunodeficiency Virus SIVmac239 Replication in Mamu-B*08-Positive Macaques

An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08⁺) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8⁺ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8⁺ T-cell epitopes to investigate the contribution of epitope-specific CD8⁺ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08⁺ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08⁺ macaques. The five most immunodominant Mamu-B*08-restricted CD8⁺ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08⁺ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08⁺ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8⁺ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08⁺ macaques.A few individuals spontaneously control the replication of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) to very low levels. The precise mechanisms underlying this control are of great interest, as a clear understanding of what constitutes a successful immune response may aid in developing an AIDS vaccine. Particularly pressing questions for vaccine design include which proteins to use as immunogens, the extent to which increasing the breadth and magnitude of responses is advantageous, how immunodomination affects T-cell responses, and if biasing the immune response toward particular effector profiles is beneficial. Characterization of immune responses made by elite controllers (ECs) may reveal patterns that can then be applied to vaccine formulation and evaluation.HIV ECs are generally not infected with grossly unfit viruses (6, 42). Instead, elite control of immunodeficiency virus replication is correlated with the presence of particular major histocompatibility complex class I (MHC-I) alleles (11, 12, 18, 32, 41, 55). The association of MHC-I alleles with the control of viremia implicates CD8⁺ T cells as being mediators of this immune containment. Several lines of evidence support this hypothesis. These lines of evidence include the correlation between the appearance of CD8⁺ T-cell responses and the resolution of peak viremia during acute infection (7, 29), the finding that alleles associated with viral control restrict dominant acute-phase CD8⁺ T-cell responses (3), and the finding that responses directed against epitopes restricted by these alleles frequently select for viral escape variants (4, 27, 38). Perhaps most compelling is the observation that for a few HIV-infected individuals, the selection of escape variants by an immunodominant HLA-B27-restricted T-cell response temporally preceded substantial increases in viremia (17, 21, 53). While viruses exhibiting escape variants in epitopes restricted by protective alleles are often detectably less fit in vitro (10, 38, 43, 51), recent data have found normal, high levels of replication in vivo upon the transmission of some of these variants (15).The association of control with MHC-I alleles does not, of course, implicate solely CD8⁺ T cells. MHC-I molecules are also ligands for killer immunoglobulin receptors (KIRs), which are predominantly expressed on natural killer (NK) cells. Genetic studies of HIV-infected humans suggest a model in which individuals with particular KIR/HLA combinations are predisposed to control HIV replication more readily than those with other KIR/HLA combinations (36, 37). These data were supported by functional studies of this KIR/HLA pairing in vitro, which demonstrated an inhibition of HIV replication by such NK cells (2). The relative contributions of NK and CD8⁺ T-cell responses to control have yet to be elucidated and may be closely intertwined.Previously, the experimental depletion of circulating CD8⁺ cells from SIVmac239-infected ECs resulted in a sharp spike in viremia, which resolved as CD8⁺ cells repopulated the periphery (19). During the reestablishment of control of SIV replication, CD8⁺ T cells targeting multiple epitopes restricted by alleles associated with elite control expanded in frequency, providing strong circumstantial evidence for their role in maintaining elite control (19, 31). However, CD8 depletion antibodies used in macaques also remove NK cells, which, at least in vitro, also inhibit SIV replication (19). It was therefore difficult to make definitive conclusions regarding the separate contributions of these subsets to maintaining the control of SIV replication in vivo.Here we investigate elite control in the rhesus macaque model for AIDS. We focused on the macaque MHC-I allele most tightly associated with the control of SIVmac239, Mamu-B*08. Approximately 50% of Mamu-B*08-positive (Mamu-B*08⁺) animals infected with SIVmac239 become ECs (32). Peptides presented by Mamu-B*08 share a binding motif with peptides presented by HLA-B27. Although these two MHC-I genes are dissimilar in domains that are important for peptide binding, each molecule can bind peptides that are presented by the other molecule (33). This striking similarity suggests that the elite control of SIVmac239 in Mamu-B*08⁺ animals is a good model for the elite control of HIV.Seven SIVmac239 epitopes restricted by Mamu-B*08 accrue variation in Mamu-B*08⁺ rhesus macaques (30, 31). For an eighth Mamu-B*08-restricted epitope, which is also restricted by Mamu-B*03 (Mamu-B*03 differs from Mamu-B*08 by 2 amino acids in the α1 and α2 domains [9, 32]), escape has been documented only for SIV-infected Mamu-B*03⁺ macaques (16). Variation in these CD8⁺ T-cell epitopes accumulates with different kinetics, starting during acute infection for those targeted by high-magnitude responses.In this study, we addressed the question of whether the elite control of SIVmac239 in Mamu-B*08⁺ animals is mediated by the known high-frequency CD8⁺ T-cell responses targeting Mamu-B*08-restricted epitopes. To this end, we introduced point mutations into eight epitopes, with the goal of reducing or abrogating immune responses directed against these epitopes during acute infection. We hypothesized that Mamu-B*08⁺ macaques would be unable to control SIV replication without these Mamu-B*08-restricted T-cell responses.