QuantiFERON-TB Gold Plus Assay in Patients With Latent vs. Active Tuberculosis in a Low Incidence Setting: Level of IFN-γ, CD4/CD8 Responses, and Release of IL-2, IP-10, and MIG.

Objectives: We analyzed the results of the QuantiFERON Glod Plus assay (QFT) and cytokine patterns associated with active tuberculosis (ATB) among patients with positive QFT. Methods: A total of 195 patients are QFT-positive, among which 24 had an ATB and 171 had a latent tuberculosis infection (LTBI). Interferon-gamma (IFN-γ) secretion was analyzed relative to interleukin-2 (IL-2), IFN-γ inducible protein or CXCL-10 (IP-10), and monokine induced by IFN-γ or CXCL-9 (MIG) secretion, and then compared between two sets of peptide antigens [tube 1 - cluster of differentiation 4 (CD4+) T cell stimulation; tube 2 - CD4+/CD8+ T cell response]. Results: Higher IFN-γ responses were measured in the ATB group (p = 0.0089). The results showed that there was a lower ratio of tube 1/tube 2 IFN-γ concentrations in the ATB group (p = 0.0009), and a median [interquartile ranges (IQR)] difference between the two sets at -0.82 IU/ml (-1.67 to 0.18) vs. -0.07 IU/ml (-0.035 to 0.11, p < 0.0001) in the ATB group compared to the LTBI group, respectively. In addition, patients with low ratios of IL-2/IFN-γ, IP-10/IFN-γ, and MIG/IFN-γ were much more likely to have ATB. Conclusion: High levels of IFN-γ secretion, preferential IFN-γ response in tube 2, and lower secretion of IL-2, IP-10, and MIG release relative to IFN-γ secretion were more likely observed in subjects with ATB. These features of T cell response may be helpful in low prevalence settings to suspect ATB in patients tested positive for IFN-γ release assays (IGRA).

Elimination of PknL and MSMEG_4242 in Mycobacterium smegmatis alters the character of the outer cell envelope and selects for mutations in Lsr2.

Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.

Mesenchymal stem cell repression of Th17 cells is triggered by mitochondrial transfer.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells with broad immunosuppressive capacities. Recently, it has been reported that MSCs can transfer mitochondria to various cell types, including fibroblast, cancer, and endothelial cells. It has been suggested that mitochondrial transfer is associated with a physiological response to cues released by damaged cells to restore and regenerate damaged tissue. However, the role of mitochondrial transfer to immune competent cells has been poorly investigated. METHODS AND RESULTS: Here, we analyzed the capacity of MSCs from the bone marrow (BM) of healthy donors (BM-MSCs) to transfer mitochondria to primary CD4+CCR6+CD45RO+ T helper 17 (Th17) cells by confocal microscopy and fluorescent-activated cell sorting (FACS). We then evaluated the Th17 cell inflammatory phenotype and bioenergetics at 4 h and 24 h of co-culture with BM-MSCs. We found that Th17 cells can take up mitochondria from BM-MSCs already after 4 h of co-culture. Moreover, IL-17 production by Th17 cells co-cultured with BM-MSCs was significantly impaired in a contact-dependent manner. This inhibition was associated with oxygen consumption increase by Th17 cells and interconversion into T regulatory cells. Finally, by co-culturing human synovial MSCs (sMSCs) from patients with rheumatoid arthritis (RA) with Th17 cells, we found that compared with healthy BM-MSCs, mitochondrial transfer to Th17 cells was impaired in RA-sMSCs. Moreover, artificial mitochondrial transfer also significantly reduced IL-17 production by Th17 cells. CONCLUSIONS: The present study brings some insights into a novel mechanism of T cell function regulation through mitochondrial transfer from stromal stem cells. The reduced mitochondrial transfer by RA-sMSCs might contribute to the persistence of chronic inflammation in RA synovitis.

Combined testing for herpes simplex virus and Mycobacterium tuberculosis DNA in cerebrospinal fluid of patients with aseptic meningitis in Burkina Faso, West Africa.

BACKGROUND: Little is known about the involvement of herpes simplex virus (HSV) or Mycobacterium tuberculosis (MTB) as potentially curable causes of central nervous system (CNS) infections in sub-Saharan Africa. OBJECTIVE: In this study, we developed a PCR assay dedicated to simultaneous testing of HSV1/HSV2 and MTB in Burkina Faso, a country where HSV is neglected as a cause of CNS infection and where TB prevalence is high. METHODS: A consensus HSV1/HSV2 set of primers and probe were designed and combined to primers and probe targeting the IS6110 repetitive insertion sequence of MTB. Analytical performances of the assay were evaluated on reference materials. Cerebrospinal fluid (CSF) collected from subjects with aseptic meningitis was tested for HSV1/HSV2 and MTB DNA. RESULTS: The UL29 gene was chosen as a highly conserved region targeted by the HSV1/HSV2 nucleic acid test. The lower limits of detection were estimated to be 2.45 copies/µL for HSV1, 1.72 copies/µL for HSV2, and 2.54 IS6110 copies per µL for MTB. The PCR was used in 202 CSF collected from subjects suspected of aseptic meningitis. Five samples (2.46%) tested positive, including two children positive for HSV1 (0.99%) and three adults tested positive for MTB (1.47%). CONCLUSION: Using an in-house real-time PCR assay, we showed that both HSV and MTB are etiologic pathogens contributing to aseptic meningitis in Burkina Faso. This molecular test may have clinical utility for early diagnosis for those treatable CNS infections.

Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens.

Background: When available, nucleic acid tests (NATs) offer powerful tools to strengthen the potential of tuberculosis (TB) diagnosis assays. The sensitivity of molecular assays is critical for detection of Mycobacterium tuberculosis (MTB) in paucibacillary sputum. Materials and Methods: The impact of targeting repetitive IS6110 sequences on the PCR sensitivity was evaluated across mycobacterium strains and reference material. Six lysis-extraction protocols were compared. Next, 92 clinical sputum specimens including 62 culture-positive samples were tested and the results were compared to sputum-smear microscopy, culture, and Xpert MTB/RIF test. Finally, the capacity to detect low MTB DNA concentrations was assessed in 40 samples containing <1.5 × 102 copies/ml ex vivo or after dilution. Results: The lower limit of detection (LOD) using the IS6110 PCR was 107 genome copies/ml (95% CI: 83-130) using MTB H37Rv as a reference strain, versus 741 genome copies/ml (95% CI: 575-1094) using the senX3 PCR. The proportion of recovered MTB DNA after lysis and extraction ranged from 35 to 82%. The Chelex® method appeared as a more efficient protocol among the six different protocols tested. The sensitivity and specificity in clinical sputum samples were 95.1% (95% CI: 90.7-99.6) and 100% (95% CI: 96.2-100.8), respectively. Among 40 samples with low MTB DNA concentration, 75% tested positive for IS6110 PCR, versus 55% using the Xpert MTB/RIF assay (p = 0.03). Conclusion: Laboratory assays based on an efficient MTB lysis and DNA extraction protocols combined with amplification of IS6110 repeat sequences appear as a sensitive diagnostic method to detect MTB DNA in sputum with low bacterial load.

High IFN-γ Release and Impaired Capacity of Multi-Cytokine Secretion in IGRA Supernatants Are Associated with Active Tuberculosis.

Interferon gamma (IFN-γ) release assays (IGRAs) detect Mycobacterium tuberculosis (Mtb) infection regardless of the active (ATB) or latent (LTBI) forms of tuberculosis (TB). In this study, Mtb-specific T cell response against region of deletion 1 (RD1) antigens were explored by a microbead multiplex assay performed in T-SPOT TB assay (T-SPOT) supernatants from 35 patients with ATB and 115 patients with LTBI. T-SPOT is positive when over 7 IFN-γ secreting cells (SC)/250 000 peripheral blood mononuclear cells (PBMC) are enumerated. However, over 100 IFN-γ SC /250 000 PBMC were more frequently observed in the ATB group compared to the LTBI group. By contrast, lower cytokine concentrations and lower cytokine productions relative to IFN-γ secretion were observed for IL 4, IL-12, TNF-α, GM-CSF, Eotaxin and IFN-α when compared to LTBI. Thus, high IFN-γ release and low cytokine secretions in relation with IFN-γ production appeared as signatures of ATB, corroborating that multicytokine Mtb-specific response against RD1 antigens reflects host capacity to contain TB reactivation. In this way, testing cytokine profile in IGRA supernatants would be helpful to improve ATB screening strategy including immunologic tests.

Deletion of a dehydratase important for intracellular growth and cording renders rough Mycobacterium abscessus avirulent.

Mycobacterium abscessus (Mabs) is a rapidly growing Mycobacterium and an emerging pathogen in humans. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence in zebrafish, which involves the formation of massive serpentine cords, abscesses, and rapid larval death. Generating a cord-deficient Mabs mutant would allow us to address the contribution of cording in the physiopathological signs of the R variant. Herein, a deletion mutant of MAB_4780, encoding a dehydratase, distinct from the ß-hydroxyacyl-ACP dehydratase HadABC complex, was constructed in the R morphotype. This mutant exhibited an alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype not only in wild-type but also in immunocompromised zebrafish embryos lacking either macrophages or neutrophils. The abolition of granuloma formation in embryos infected with the dehydratase mutant was associated with a failure to replicate in macrophages, presumably due to limited inhibition of the phagolysosomal fusion. Overall, these results indicate that MAB_4780 is required for Mabs to successfully establish acute and lethal infections. Therefore, targeting MAB_4780 may represent an attractive antivirulence strategy to control Mabs infections, refractory to most standard chemotherapeutic interventions. The combination of a dehydratase assay with a high-resolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors.

A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence of Mycobacterium marinum in multiple hosts.

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C-terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail-forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane-defined vacuoles. Functional complementation studies indicated that the C-terminus, but not the N-terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non-pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis.

A new dehydratase conferring innate resistance to thiacetazone and intra-amoebal survival of Mycobacterium smegmatis.

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second-line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon-mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100-fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot-dog domains characteristic of dehydratase-like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis-Acanthamoeba co-cultures, we found that intra-amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.

Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.

The Mycobacterium tuberculosis transcriptional repressor EthR is negatively regulated by Serine/Threonine phosphorylation.

Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb.

7-chloroquinoline-isatin conjugates: antimalarial, antitubercular, and cytotoxic evaluation.

A series of twenty piperazine-tethered 7-chloroquinoline-isatin hybrids have been synthesized via either direct nucleophilic substitution or Cu(Ι)Cl-mediated Mannich reaction. These new conjugates were evaluated for their antimalarial and antitubercular efficacy against a chloroquine-resistant strain of Plasmodium falciparum and Mycobacterium tuberculosis, respectively, while the cytotoxic profiles were evaluated against 3T6 cell line, a permanent mouse embryonic fibroblast cell line. The most potent of the test compound with IC50 of 0.22 µm against W2 strain of P. falciparum and 31.62 µm against the embryonic fibroblast cell line (cytotoxicity) displayed a high selective index of 143.73.

4-Aminoquinoline-ß-lactam conjugates: synthesis, antimalarial, and antitubercular evaluation.

A library of quinoline-ß-lactam-based hybrids was synthesized and tested for their antimalarial and antitubercular activities. The present antimalarial data showed the dependence of activity on the nature of linker, N-1 substituent of the ß-lactam ring as well as the length of alkyl chain. Most of the compounds are not as efficient as chloroquine in inhibiting the culture growth of Plasmodium falciparum W2 strain. Nevertheless, the synthesized hybrids showed better antitubercular activities (up to five times) compared with cephalexin (up to three times) and ethionamide.

Overexpression of the KdpF membrane peptide in Mycobacterium bovis BCG results in reduced intramacrophage growth and altered cording morphology.

Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, such as sensor kinases. To date, regulatory membrane peptides have been completely overlooked in mycobacteria. The 30 amino-acid-long KdpF peptide, which is co-transcribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s) towards the KdpD sensor kinase. Herein, we showed by quantitative RT-PCR that the Mycobacterium bovis BCG kdpAB and kdpDE genes clusters are differentially induced in potassium-deprived broth medium or within infected macrophages. We have overexpressed the kdpF gene in M. bovis BCG to investigate its possible regulatory role and effect on mycobacterial virulence. Our results indicate that KdpF does not play a critical regulatory role on kdp genes expression despite the fact that KdpF interacts with the KdpD sensor kinase in a bacterial two-hybrid assay. However, overexpression of kdpF results in a significant reduction of M. bovis BCG growth in both murine and human primary macrophages, and is associated with a strong alteration of colonial morphology and impaired cording formation. To identify novel KdpF interactants, a mycobacterial library was screened using KdpF as bait in the bacterial two-hybrid system. This allowed us to identify members of the MmpL family of membrane proteins, known to participate in the biosynthesis/transport of various cell wall lipids, thus highlighting a possible link between KdpF and cell wall lipid metabolism. Taken together, these data suggest that KdpF overexpression reduces intramacrophage growth which may result from alteration of the mycobacterial cell wall.

Azide-alkyne cycloaddition en route towards 1H-1,2,3-triazole-tethered ß-lactam-ferrocene and ß-lactam-ferrocenylchalcone conjugates: synthesis and in vitro anti-tubercular evaluation.

A diverse range of triazoles were prepared following well established, Cu-mediated azide-alkyne cycloaddition reactions with the aim of probing the anti-tubercular structure-activity relationships (SAR) within the ß-lactam-ferrocene-triazole conjugate family. The anti-tubercular evaluation studies of the synthesized conjugates revealed that none of the scaffolds exhibited any activity that restricted mycobacterial growth even at high doses. The introduction of various substituents onto the N-1 of the ß-lactam ring, introducing mono- or di-ferrocenylchalcone substituents at the C-3 position as well as introducing a spacer of varying chain length failed to produce any significant enhancement in the activity profiles. The described protocol was a successful attempt on the inclusion of a ferrocene nucleus in the ß-lactam family tethered via triazole linkers having metabolic stability and physicochemical favourability.

The C-terminal domain of the virulence factor MgtC is a divergent ACT domain.

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg(2+) deprivation, but previous work suggested that MgtC is not a Mg(2+) transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg(2+) deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg(2+) concentration, indicating that it does not bind Mg(2+). The structure of the C-terminal domain forms a ßαßßαß fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg(2+) uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.

Fatty acyl chains of Mycobacterium marinum lipooligosaccharides: structure, localization and acylation by PapA4 (MMAR_2343) protein.

We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR_2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR_2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR_2343 gene. Disruption of MMAR_2343 resulted in a severe LOS breakdown, indicating that MMAR_2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly.

Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions.

The hepatitis C virus genome encodes a polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases to yield 10 mature protein products (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Although most cleavages in hepatitis C virus polyprotein precursor proceed to completion during or immediately after translation, the cleavages mediated by a host cell signal peptidase are partial at the E2/p7 and p7/NS2 sites, leading to the production of an E2p7NS2 precursor. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can function as signal peptides. When fused to a reporter protein, the signal peptides of p7 and NS2 were efficiently cleaved. However, when full-length p7 was fused to the reporter protein, partial cleavage was observed, indicating that a sequence located N-terminally of the signal peptide reduces the efficiency of p7/NS2 cleavage. Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both the E2/p7 and p7/NS2 sites. Finally, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane alpha-helix within the P' region might impose additional structural constraints to the cleavage sites. The insertion of a linker polypeptide sequence between P-3' and P-4' of the cleavage site released these constraints and led to improved cleavage efficiency. Such constraints in the processing of a polyprotein precursor are likely essential for hepatitis C virus to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.

Subcellular localization and topology of the p7 polypeptide of hepatitis C virus.

Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.