A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Host Cell-Virus Cross Talk: Phosphorylation of a Hepatitis B Virus Envelope Protein Mediates Intracellular Signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S→A) or mimicking (S→D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

Vaccinia Virus H7 Protein Contributes to the Formation of Crescent Membrane Precursors of Immature Virions

Crescent membranes are the first viral structures that can be discerned during poxvirus morphogenesis. The crescents consist of a lipoprotein membrane and an outer lattice scaffold, which provides uniform curvature. Relatively little is known regarding the composition of the crescent membrane or its mode of formation. Here, we show that the H7 protein, which is conserved in all vertebrate poxviruses but has no discernible functional motifs or nonpoxvirus homologs, contributes to the formation of crescents and immature virions. Synthesis of the 17-kDa H7 protein was dependent on DNA replication and occurred late during vaccinia virus infection. Unlike many late proteins, however, H7 was not incorporated into mature virions or localized in cellular organelles. To gain insight into the role of H7, an inducible mutant was constructed and shown to have a conditional lethal phenotype: H7 expression and infectious virus formation were dependent on isopropyl-beta-d-thiogalactopyranoside. In the absence of inducer, viral late proteins were made, but membrane and core proteins were not processed by the I7 protease. A block in morphogenesis was demonstrated by transmission electron microscopy: neither typical crescents nor immature virions were detected in the absence of inducer. Instead, factory areas of the cytoplasm contained large, electron-dense inclusions, some of which had partially coated membrane segments at their surfaces. Separate, lower-density inclusions containing the D13 scaffold protein and endoplasmic reticulum membranes were also present. These features are most similar to those previously seen when expression of A11, another conserved nonvirion protein, is repressed.The vertebrate poxviruses, of which vaccinia virus (VACV) is the prototype, encode about 200 proteins, of which almost half are conserved in all species (40). The conserved proteins include those that execute basic functions, which allow poxviruses to replicate and express their double-stranded DNA genomes and assemble infectious particles in the cytoplasm (25). Due to their large number, some of the conserved open reading frames (ORFs) have yet to be characterized. In the present study, we show that the product of the VACV H7R ORF contributes to the formation of the crescent membrane precursors of immature virions (IVs).Crescents are uniformly curved membranes that form within specialized regions of the cytoplasm known as factories (8, 10). The crescents envelop electron-dense granular material containing core precursor proteins to form ∼300-nm spherical IVs, which subsequently undergo internal and external architectural changes to become infectious brick-shaped mature virions (MVs) (6). Several models have been proposed for the structure and mode of formation of crescents and IVs. Transmission electron micrography revealed a single membrane bilayer covered with an external “spicule” coat (8, 24). Although evidence for two closely apposed membranes has been presented (29, 34), other studies support the original single membrane structure (5, 17-19). The outer coat was revealed by deep-etch immunogold electron microscopy to be a lattice comprised of trimers of the D13 protein (18, 36) rather than a layer of discontinuous spikes.The crescent and IV membranes are not fully characterized with regard to their composition or organization, and two main theories regarding their origin have been proposed. One idea, inspired by the spatial separation of crescent and cellular membranes in virus factories, was the de novo origin of poxvirus membranes from lipids and viral proteins (9). An alternative model, positing the derivation of crescents from cellular membranes, was based partly on the lack of precedence of de novo membrane formation in other biological systems, the finding of some viral proteins associated with membranes of the intermediate compartment between the endoplasmic reticulum (ER) and the Golgi apparatus, and the proximity of tubular structures and viral membranes (29, 33). Other studies, however, provided evidence for trafficking of proteins to the viral membrane through the ER rather than the intermediate compartment although the initial membrane nucleation event was not investigated (20, 21).Understanding the mechanism of viral membrane formation depends on the identification of the viral and cellular proteins involved. A role for the cellular coatomer and KDEL receptor in early VACV biogenesis has been suggested (43). Studies of conditional lethal VACV mutants pointed to the involvement of several viral proteins in the formation of crescent membranes. Repression of synthesis of the D13 scaffold protein mimics the effect of the drug rifampin and results in floppy-appearing membranes bordering electron-dense granular material (44). Such membranes seem otherwise normal as they can acquire the scaffold and concomitant rigid curvature within minutes after removal of rifampin and develop into IVs (26). Repression of synthesis of the integral membrane proteins A14 and A17 results in aberrant vesicular or tubular structures that differ from each other in appearance (30, 31, 39, 41). Both A14 and A17 are phosphorylated by the F10 kinase (3, 13, 39), and viral membranes are not detected when cells are infected with conditional lethal F10 mutants under nonpermissive conditions (37, 38). Viral membranes are also not observed under nonpermissive conditions when cells are infected with conditional lethal H5 (12), G5 (7), and A11 (28) mutants though their roles in this process are not yet understood. Here, we characterize the product of the H7R ORF and demonstrate that it is also involved in viral membrane formation and morphogenesis.     

Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.     

Identification of Functional Domains in the 14-Kilodalton Envelope Protein (A27L) of Vaccinia Virus

The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126-10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.     

Oversulfated Chondroitin Sulfate Binds to Chemokines and Inhibits Stromal Cell-Derived Factor-1 Mediated Signaling in Activated T Cells

Oversulfated chondroitin sulfate (OSCS), a member of the glycosaminoglycan (GAG) family, was a contaminant in heparin that was linked to the 2008 heparin adverse events in the US. Because of its highly negative charge, OSCS can interact with many components of the contact and immune systems. We have previously demonstrated that OSCS inhibited the complement classical pathway by binding C1 inhibitor and potentiating its interaction with C1s. In the present study, by using surface plasmon resonance, we found OSCS interacts with T cell chemokines that can impact adaptive immunity. The binding of OSCS to stromal cell-derived factor-1 (SDF-1) chemokines, SDF-1α and SDF-1β, caused a significant change in the secondary structures of these chemokines as detected by far-ultraviolet circular dichroism spectra analysis. Functionally, OSCS binding profoundly inhibited SDF-1-induced calcium mobilization and T cell chemotaxis. Imaging flow cytometry revealed T cell morphological changes mediated by SDF-1α were completely blocked by OSCS. We conclude that the OSCS, a past contaminant in heparin, has broad interactions with the components of the human immune system beyond the contact and complement systems, and that may explain, in part, prior OSCS-related adverse events, while suggesting potentially useful therapeutic applications for related GAGs in the control of inflammation.     

Antigenic Properties of the Human Immunodeficiency Virus Envelope Glycoprotein Gp120 on Virions Bound to Target Cells

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.     

Binding of Sindbis Virus to Cell Surface Heparan Sulfate

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.     

Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

Our previous studies demonstrated that the cell culture-grown hepatitis C virus of genotype 2a (HCVcc) uses apolipoprotein E (apoE) to mediate its attachment to the surface of human hepatoma Huh-7.5 cells. ApoE mediates HCV attachment by binding to the cell surface heparan sulfate (HS) which is covalently attached to the core proteins of proteoglycans (HSPGs). In the present study, we further determined the physiological importance of apoE and HSPGs in the HCV attachment using a clinical HCV of genotype 1b (HCV1b) obtained from hepatitis C patients and human embryonic stem cell-differentiated hepatocyte-like cells (DHHs). DHHs were found to resemble primary human hepatocytes. Similar to HCVcc, HCV1b was found to attach to the surface of DHHs by the apoE-mediated binding to the cell surface HSPGs. The apoE-specific monoclonal antibody, purified HSPGs, and heparin were all able to efficiently block HCV1b attachment to DHHs. Similarly, the removal of heparan sulfate from cell surface by treatment with heparinase suppressed HCV1b attachment to DHHs. More significantly, HCV1b attachment was potently inhibited by a synthetic peptide derived from the apoE receptor-binding region as well as by an HSPG-binding peptide. Likewise, the HSPG-binding peptide prevented apoE from binding to heparin in a dose-dependent manner, as determined by an in vitro heparin pull-down assay. Collectively, these findings demonstrate that HSPGs serve as major HCV attachment receptors on the surface of human hepatocytes to which the apoE protein ligand on the HCV envelope binds.     

Envelope Formation Is Blocked by Mutation of a Sequence Related to the HKD Phospholipid Metabolism Motif in the Vaccinia Virus F13L Protein

The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus.     

Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

Poxviruses produce complement regulatory proteins to subvert the host''s immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host''s immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement''s role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE''s regulatory capacity. These results suggest that EMICE''s role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.Poxviruses encode in their large double-stranded DNA genomes many factors that modify the immune system (30, 56). The analysis of these molecules has revealed a delicate balance between viral pathogenesis and the host''s immune response (2, 21, 31, 61). Variola, vaccinia, monkeypox, cowpox, and ectromelia (ECTV) viruses each produce an orthologous complement regulatory protein (poxviral inhibitor of complement enzymes [PICE]) that has structural and functional homology to host proteins (14, 29, 34, 38, 41, 45, 54). The loss of the regulatory protein resulted in smaller local lesions with vaccinia virus lacking the vaccinia virus complement control protein (VCP) (29) and in a greater local inflammatory response in the case of cowpox lacking the inflammation-modulatory protein (IMP; the cowpox virus PICE) (35, 45, 46). Additionally, the complete loss of the monkeypox virus inhibitor of complement enzymes (MOPICE) may account for part of the reduced mortality observed in the West African compared to Congo basin strains of monkeypox virus (12).The complement system consists of proteins on the cell surface and in blood that recognize and destroy invading pathogens and infected host cells (36, 52). Viruses protect themselves from the antiviral effects of complement activation in a variety of ways, including hijacking the host''s complement regulatory proteins or producing their own inhibitors (7, 8, 15, 20, 23). Another effective strategy is to incorporate the host''s complement regulators in the outermost viral membrane, which then protects the virus from complement attack (62). The extracellular enveloped virus (EEV) produced by poxviruses acquires a unique outer membrane derived from the Golgi complex or early endosomes that contain the protective host complement regulators (58, 62). Poxviruses have multiple infectious forms, and the most abundant, intracellular mature virions (IMV), are released when infected cells lyse (58). The IMV lacks the outermost membrane found on EEV and is sensitive to complement-mediated neutralization. The multiple strategies viruses have evolved to evade the complement system underscore its importance to innate and adaptive immunity (15, 36).The most well-characterized PICE is VCP (24-29, 34, 49, 50, 53, 55, 59, 60). Originally described as a secreted complement inhibitor (34), VCP also attaches to the surface of infected cells through an interaction with the viral membrane protein A56 that requires an unpaired N-terminal cysteine (26). This extra cysteine also adds to the potency of the inhibitor by forming function-enhancing dimers (41). VCP and the smallpox virus inhibitor of complement enzymes (SPICE) bind heparin in vitro, and this may facilitate cell surface interactions (24, 38, 50, 59). The coevolution of variola virus with its only natural host, humans, likely explains the enhanced activity against human complement observed with SPICE compared to the other PICEs (54, 64).Our recent work with ECTV, the causative agent of mousepox infection, demonstrated that the classical and alternative pathways of the complement system are required for host survival (48). The mouse-specific pathogen ECTV causes severe disease in most strains and has coevolved with its natural host, analogous to variola virus in humans (9). This close host-virus relationship is particularly important for evaluating the role of the complement system, given the species specificity of many complement proteins, receptors, and regulators (10, 47, 62). Additionally, the availability of complement-deficient mice permits dissection of the complement activation pathways involved. Naïve C57BL/6 mouse serum neutralizes the IMV of ECTV in vitro, predominately through opsonization (48). Maximal neutralization requires natural antibody, classical-pathway activation, and amplification by the alternative pathway. C3 deficiency in the normally resistant C57BL/6 strain results in acute mortality, similar to immunodeficiencies in important elements of the antiviral immune response, including CD8⁺ T cells (19, 32), natural killer cells (18, 51), and gamma interferon (33). During ECTV infection, the complement system acts in the first few hours and days to delay the spread of infection, resulting in lower levels of viremia and viral burden in tissues (48).This study characterized the PICE produced by ECTV, ectromelia virus inhibitor of complement enzymes (EMICE), and assessed its complement regulatory activity. Recombinant EMICE (rEMICE) decreased activation of both human and mouse complement. Murine cells produced EMICE at 4 to 6 h postinfection prior to the release of the majority of the complement-sensitive IMV from infected cells. rEMICE protected ECTV IMV from complement-mediated neutralization. Further, EMICE produced during natural infection inhibited complement deposition on infected cells by the alternative pathway. ECTV likely produces this abundance of EMICE to protect both the IMV and infected cells.     

Down Regulation of Gene Expression by the Vaccinia Virus D10 Protein

Vaccinia virus genes are expressed in a sequential fashion, suggesting a role for negative as well as positive regulatory mechanisms. A potential down regulator of gene expression was mapped by transfection assays to vaccinia virus open reading frame D10, which encodes a protein with no previously known function. Inhibition was independent of the promoter type used for the reporter gene, indicating that the mechanism did not involve promoter sequence recognition. The inhibition was overcome, however, when the open reading frame of the reporter gene was preceded by the encephalomyocarditis virus internal ribosome entry site, which excludes the possibility of nonspecific metabolic or other antiviral effects and suggests that capped mRNAs or cap-dependent translation might be the target of the D10 product. The inducible overexpression of the D10 gene by a recombinant vaccinia virus severely inhibited viral protein synthesis, decreased the steady-state level of viral late mRNA, and blocked the formation of infectious virus.     

Chondroitin Sulfate as a Molecular Portal That Preferentially Mediates the Apoptotic Killing of Tumor Cells by Penetratin-directed Mitochondria-disrupting Peptides

The use of cell-penetrating peptides (CPPs) as drug carriers for targeted therapy is limited by the unrestricted cellular translocation of CPPs. The preferential induction of tumor cell death by penetratin (Antp)-directed peptides (PNC27 and PNC28), however, suggests that the CPP Antp may contribute to the preferential cytotoxicity of these peptides. Using PNC27 as a molecular model, we constructed three novel peptides (PT, PR9, and PD3) by replacing the leader peptide Antp with one of three distinct CPPs (TAT, R9, or DPV3), respectively. The IC₅₀ values of PNC27 in tumor cells were 2–3 times lower than in normal cells. However, all three engineered peptides demonstrated similar cytotoxic effects in tumor and normal cells. Another three chimeric peptides containing the leader peptide Antp with different mitochondria-disrupting peptides (KLA-Antp (KGA), B27-Antp (BA27), and B28-Antp (BA28)), preferentially induced apoptosis in tumor cells. The IC₅₀ values of these peptides (3–10 μm) were 3–6 times lower in tumor cells than in normal cells. In contrast, TAT-directed peptides (TAT-KLA (TK), TAT-B27 (TB27), and TAT-B28 (TB28)), were cytotoxic to both tumor and normal cells. These data demonstrate that the leader peptide Antp contributes to the preferential cytotoxicity of Antp-directed peptides. Furthermore, Antp-directed peptides bind chondroitin sulfate (CS), and the removal of endogenous CS reduces the cytotoxic effects of Antp-directed peptides in tumor cells. The overexpression of CS in tumor cells is positively correlated to the cell entry and cytotoxicity of Antp- directed peptides. These results suggest that CS overexpression in tumor cells is an important molecular portal that mediates the preferential cytotoxicity of Antp-directed peptides.     

丙型肝炎病毒E2蛋白对HepG2细胞MAPK／ERK的激活

人CD81是丙型肝炎病毒（hepatitis Cvirus,HCV）的细胞表面特异性受体,HCV包膜蛋白－2（E2）可与其结合。细胞个信号调节激酶（extracellular signal-regulated protein kinase,MAPK/ERK1,2）信号途径主要介导细胞增殖及分化。为探讨HCV E2蛋白与人CD81结合对MAPK／ERK活性变化的影响,以HCV E2蛋白刺激HepG2细胞,采用免疫印迹、免疫组化及免疫荧光等方法动态观察细胞内MAPK／ERK的激活情况,并以流式细胞术检测细胞表面CD81的表达。结果表明：HepG2细胞高表达人CD81;HCV E2蛋白可激活细胞内MAPK／ERK;MAPK／ERK的磷酸化反应与HCV E2蛋白浓度、作用时间呈依赖关系;HCV E2－CD81相互作用引发的细胞异常信号转导可能与HCV致病性相关。