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1.
Liu C Feng Y Gao F Zhang Q Wang M 《Biochemical and biophysical research communications》2006,345(3):1108-1115
Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six alpha-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection. 相似文献
2.
Zhu J Zhang CW Qi Y Tien P Gao GF 《Biochemical and biophysical research communications》2002,299(5):897-902
Recent studies have shown that paramyxovirus might adopt a similar molecular mechanism of virus entry and fusion in which the attachment glycoprotein binds receptor/s and triggers the conformational changes of the fusion protein. There are two conserved regions of heptad repeat (HR1 and HR2) in the fusion protein and they were shown with fusion-inhibition effects in many paramyxoviruses, including measles virus. They also appear to show characteristic structure in the fusion core: the HR1/HR2 forms stable six-helix coiled-coil centered by HR1 and is surrounded by HR2 (trimer of HR1/HR2), which represents the post-fusion conformational structure. In this study, we expressed the HR1 and HR2 of measles virus fusion protein as a single chain (named 2-Helix) and subsequently tested its formation of trimer. Indeed, the results do show that the HR1 and HR2 interact with each other and form stable six-helix coiled-coil bundle. This is the first member in genus Morbillivirus of family Paramyxoviridae to be confirmed with this characteristic structure and provides the basis for the HR2-inhibition effects on virus fusion/entry for measles virus. 相似文献
3.
Xu Y Cole DK Lou Z Liu Y Qin L Li X Bai Z Yuan F Rao Z Gao GF 《Protein expression and purification》2004,38(1):116-122
Membrane fusion between virus and host cells is the key step for enveloped virus entry and is mediated by the viral envelope fusion protein. In murine coronavirus, mouse hepatitis virus (MHV), the spike (S) protein mediates this process. Recently, the formation of anti-parallel 6-helix bundle of the MHV S protein heptad repeat (HR) regions (HR1 and HR2) has been confirmed, implying coronavirus has a class I fusion protein. This bundle is also called fusion core. To facilitate the solution of the crystal structure of this fusion core, we deployed an Escherichia coli in vitro expression system to express the HR1 and HR2 regions linked together by a flexible linker as a single chain (named 2-helix). This 2-helix polypeptide subsequently assembled into a typical 6-helix bundle. This bundle has been analyzed by a series of biophysical and biochemical techniques and confirmed that the design technique can be used for coronavirus as we successfully used for members of paramyxoviruses. 相似文献
4.
The fusion protein of enveloped viruses mediates the fusion between the viral and cellular membranes, allowing the penetration of the viral genomes into the host cell. Many of these proteins share a common fold comprising a central core trimer of anti-parallel coiled-coil heterodimers, which are formed by two discontinuous heptad repeat (HR) motifs located at the ectodomain of the fusion proteins. In this study, we constructed and purified the corresponding regions (HR1 and HR2) of mumps virus fusion protein that are predicted to form coiled coil. The HR1 and HR2 were expressed and purified separately or as a single chain connected by an amino acid linker (HR1-linker-HR2, named 2-Helix). Series of biochemical and biophysical analyses of the expressed proteins have shown that HR1 and HR2 of mumps virus fusion protein share the common features of other enveloped virus fusion proteins. CD spectral results show that HR1 forms an alpha-helical coil structure while HR2 exists as an unstructured monomer in PBS in nature. Mixtures of HR1 and HR2 could form a stable six-helix bundle, indicating the interaction of HR1 and HR2. The 2-Helix protein also shows characteristic properties of the 6-helix bundle. Therefore, mumps virus fusion protein has a common core architecture and its HR regions could be used as a drug target for virus fusion inhibitors. 相似文献
5.
Xu Y Zhu J Liu Y Lou Z Yuan F Liu Y Cole DK Ni L Su N Qin L Li X Bai Z Bell JI Pang H Tien P Gao GF Rao Z 《Biochemistry》2004,43(44):14064-14071
Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emergent virus responsible for a worldwide epidemic in 2003. The coronavirus spike proteins belong to class I fusion proteins, and are characterized by the existence of two heptad repeat (HR) regions, HR1 and HR2. The HR1 region in coronaviruses is predicted to be considerably longer than that in other type I virus fusion proteins. Therefore the exact binding sequence to HR2 from the HR1 is not clear. In this study, we defined the region of HR1 that binds to HR2 by a series of biochemical and biophysical measures. Subsequently the defined HR1 (902-952) and HR2 (1145-1184) chains, which are different from previously defined binding regions, were linked together by a flexible linker to form a single-chain construct, 2-Helix. This protein was expressed in Escherichia coli and forms a typical six-helix coiled coil bundle. Highly conserved HR regions between mouse hepatitis virus (MHV) and SARS-CoV spike proteins suggest a similar three-dimensional structure for the two fusion cores. Here, we constructed a homology model for SARS coronavirus fusion core based on our biochemical analysis and determined the MHV fusion core structure. We also propose an important target site for fusion inhibitor design and several strategies, which have been successfully used in fusion inhibitor design for human immunodeficiency virus (HIV), for the treatment of SARS infection. 相似文献
6.
Jing Gao Guangwen Lu Jianxun Qi Yan Li Ying Wu Yao Deng Heyuan Geng Hongbin Li Qihui Wang Haixia Xiao Wenjie Tan Jinghua Yan George F. Gao 《Journal of virology》2013,87(24):13134-13140
Middle East respiratory syndrome coronavirus (MERS-CoV) recently emerged as a severe worldwide public health concern. The virus is highly pathogenic, manifesting in infected patients with an approximately 50% fatality rate. It is known that the surface spike (S) proteins of coronaviruses mediate receptor recognition and membrane fusion, thereby playing an indispensable role in initiating infection. In this process, heptad repeats 1 and 2 (HR1 and HR2) of the S protein assemble into a complex called the fusion core, which represents a key membrane fusion architecture. To date, however, the MERS-CoV fusion core remains uncharacterized. In this study, we performed a series of biochemical and biophysical analyses characterizing the HR1/HR2 complexes of this novel virus. The HR sequences were variably truncated and then connected with a flexible amino acid linker. In each case, the recombinant protein automatically assembled into a trimer in solution, displaying a typical α-helical structure. One of these trimers was successfully crystallized, and its structure was solved at a resolution of 1.9 Å. A canonical 6-helix bundle, like those reported for other coronaviruses, was revealed, with three HR1 helices forming the central coiled-coil core and three HR2 chains surrounding the core in the HR1 side grooves. This demonstrates that MERS-CoV utilizes a mechanism similar to those of other class I enveloped viruses for membrane fusion. With this notion, we further identified an HR2-based peptide that could potently inhibit MERS-CoV fusion and entry by using a pseudotyped-virus system. These results lay the groundwork for future inhibitory peptidic drug design. 相似文献
7.
Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core 总被引:10,自引:0,他引:10
Xu Y Lou Z Liu Y Pang H Tien P Gao GF Rao Z 《The Journal of biological chemistry》2004,279(47):49414-49419
Severe acute respiratory syndrome coronavirus is a newly emergent virus responsible for a recent outbreak of an atypical pneumonia. The coronavirus spike protein, an enveloped glycoprotein essential for viral entry, belongs to the class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions are understood to form a fusion-active conformation similar to those of other typical viral fusion proteins. This hairpin structure likely juxtaposes the viral and cellular membranes, thus facilitating membrane fusion and subsequent viral entry. The fusion core protein of severe acute respiratory syndrome coronavirus spike protein was crystallized, and the structure was determined at 2.8 A of resolution. The fusion core is a six-helix bundle with three HR2 helices packed against the hydrophobic grooves on the surface of central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. This structure shares significant similarity with the fusion core structure of mouse hepatitis virus spike protein and other viral fusion proteins, suggesting a conserved mechanism of membrane fusion. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation, which have been successfully used in human immunodeficiency virus 1 inhibitor development, may be applicable to the inhibition of severe acute respiratory syndrome coronavirus on the basis of structural information provided here. The relatively deep grooves on the surface of the central coiled coil will be a good target site for the design of viral fusion inhibitors. 相似文献
8.
Xu Y Liu Y Lou Z Qin L Li X Bai Z Pang H Tien P Gao GF Rao Z 《The Journal of biological chemistry》2004,279(29):30514-30522
The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome. 相似文献
9.
Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion 总被引:6,自引:0,他引:6
Wang E Sun X Qian Y Zhao L Tien P Gao GF 《Biochemical and biophysical research communications》2003,302(3):469-475
Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins. They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV. Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus. However, in other members, only HR2 gives strong inhibition whereas HR1 does not. Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately. Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known. In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed. It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form. 相似文献
10.
Recombinant protein containing one heptad-repeat 1 (HR1) segment and one HR2 segment of the HIV-1 gp41 (HR1-HR2) has been shown to fold into thermally stable six-helix bundle, representing the fusogenic core of gp41. In this study, we have used the fusogenic core as a scaffold to design HIV-1 fusion inhibitory proteins by linking another HR1 to the C terminus of HR1-HR2 (HR121) or additional HR2 to the N terminus of HR1-HR2 (HR212). Both recombinant proteins could be abundantly and solubly expressed and easily purified, exhibiting high stability and potent inhibitory activity on HIV-1 fusion with IC50 values of 16.2+/-2.8 and 2.8+/-0.63 nM, respectively. These suggest that these rationally designed proteins can be further developed as novel anti-HIV-1 therapeutics. 相似文献
11.
Liu Y Xu Y Lou Z Zhu J Hu X Gao GF Qiu B Rao Z Tien P 《Biochemical and biophysical research communications》2006,348(3):916-922
The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar characterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins. 相似文献
12.
Hakansson-McReynolds S Jiang S Rong L Caffrey M 《The Journal of biological chemistry》2006,281(17):11965-11971
The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS. 相似文献
13.
Examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region
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Paramyxoviruses are a leading cause of childhood illness worldwide. A recently discovered paramyxovirus, human metapneumovirus (hMPV), has been studied by our group in order to determine the structural relevance of its fusion (F) protein to other well-characterized viruses utilizing type I integral membrane proteins as fusion aids. Sequence analysis and homology models suggested the presence of requisite heptad repeat (HR) regions. Synthetic peptides from HR regions 1 and 2 (HR-1 and -2, respectively) were induced to form a thermostable (melting temperature, approximately 90 degrees C) helical structure consistent in mass with a hexameric coiled coil. Inhibitory studies of hMPV HR-1 and -2 indicated that the synthetic HR-1 peptide was a significant fusion inhibitor with a 50% inhibitory concentration and a 50% effective concentration of approximately 50 nM. Many viral fusion proteins are type I integral membrane proteins utilizing the formation of a hexameric coiled coil of HR peptides as a major driving force for fusion. Our studies provide evidence that hMPV also uses a coiled-coil structure as a major player in the fusion process. Additionally, viral HR-1 peptide sequences may need further investigation as potent fusion inhibitors. 相似文献
14.
Dwyer JJ Wilson KL Martin K Seedorff JE Hasan A Medinas RJ Davison DK Feese MD Richter HT Kim H Matthews TJ Delmedico MK 《Protein science : a publication of the Protein Society》2008,17(4):633-643
HIV fusion is mediated by a conformational transition in which the C-terminal region (HR2) of gp41 interacts with the N-terminal region (HR1) to form a six-helix bundle. Peptides derived from the HR1 form a well-characterized, trimeric coiled-coil bundle in the presence of HR2 peptides, but there is little structural information on the isolated HR1 trimer. Using protein design, we have designed synthetic HR1 peptides that form soluble, thermostable HR1 trimers. In vitro binding of HR2 peptides to the engineered trimer suggests that the design strategy has not significantly impacted the ability to form the six-helix bundle. The peptides have enhanced antiviral activity compared to wild type, with up to 30-fold greater potency against certain viral isolates. In vitro passaging was used to generate HR1-resistant virus and the observed resistance mutations map to the HR2 region of gp41, demonstrating that the peptides block the fusion process by binding to the viral HR2 domain. Interestingly, the activity of the HR2 fusion inhibitor, enfuvirtide (ENF), against these resistant viruses is maintained or improved up to fivefold. The 1.5 A crystal structure of one of these designs has been determined, and we show that the isolated HR1 is very similar to the conformation of the HR1 in the six-helix bundle. These results provide an initial model of the pre-fusogenic state, are attractive starting points for identifying novel fusion inhibitors, and offer new opportunities for developing HIV therapeutics based on HR1 peptides. 相似文献
15.
Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus. 相似文献
16.
Organization of Two Transmissible Gastroenteritis Coronavirus Membrane Protein Topologies within the Virion and Core 总被引:6,自引:0,他引:6
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David Escors Emilio Camafeita Javier Ortego Hubert Laude Luis Enjuanes 《Journal of virology》2001,75(24):12228-12240
The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of (35)S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification. 相似文献
17.
Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (K(3) approximately 2.2 x 10(11) M(-2)) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with beta-sheet-like characteristics. Only a small increase in alpha-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999). Finally, HR3 forms weak alpha-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture. 相似文献
18.
We describe the properties of the two heptad repeats (HR1 and HR2) of the Peste des petits ruminants virus (PPRV) fusion protein (F) to obtain insights into the mechanism by which these repeats influence PPRV-mediated cell fusion. Both HR1 and HR2 inhibit PPRV-mediated syncytia formation in Vero cells in vitro. Of these, HR2 was found to be more effective than HR1. We studied the mechanism of fusion inhibition by these two repeats by using various biophysical and biochemical methods either separately or together. CD spectral analysis of these repeats revealed that the alpha-helical content of HR1 and HR2 when used together is higher than that of their simulated spectrum in the mixture, suggesting the formation of a highly structured complex by these repeats. Protease protection assays confirmed that such a complex is highly stable. Electrospray mass spectrometry of protease-digested products of the HR1-HR2 complex showed protection of fragments corresponding to both HR1 and HR2 sequences involved in complex formation. By employing size-exclusion chromatography and chemical cross-linking experiments, we show that three units each of HR1 and HR2 form a complex in which HR1 is a trimer and HR2 is a monomer. Homology-based three-dimensional modeling of this complex showed that HR1 and HR2 together form a six-helix and trimeric coiled-coil bundle. In this model, the HR1 trimer forms the core whereas HR2, while interacting with HR1 in an antiparallel orientation, forms a two-stranded coiled-coil structure and lies at the periphery of the structure. These results are discussed in the context of a common fusion mechanism among paramyxoviruses. 相似文献
19.
The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex 总被引:17,自引:0,他引:17
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Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein. 相似文献
20.
Design and characterization of viral polypeptide inhibitors targeting Newcastle disease virus fusion
Paramyxovirus infections can be detected worldwide with some emerging zoonotic viruses and currently there are no specific therapeutic treatments or vaccines available for many of these diseases. Recent studies have demonstrated that peptides derived from the two heptad repeat regions (HR1 and HR2) of paramyxovirus fusion proteins could be used as inhibitors of virus fusion. The mechanism underlying this activity is in accordance with that of class I virus fusion proteins, of which human immunodeficiency virus (HIV) and influenza virus fusion proteins are members. For class I virus fusion proteins, the HR1 fragment binds to HR2 to form a six-helix bundle with three HR1 fragments forming the central coiled bundle surrounded by three coiled HR2 fragments in the post fusion conformational state (fusion core). It is hypothesized that the introduced exogenous HR1 or HR2 can compete against their endogenous counterparts, which results in fusion inhibition. Using Newcastle disease virus (NDV) as a model, we designed several protein inhibitors, denoted HR212 as well asHR121 and 5-Helix, which could bind the HR1 or HR2 region of fusion protein, respectively. All the proteins were expressed and purified using a GST-fusion expression system in Escherichia coli. The HR212 or GST-HR212 protein, which binds the HR1 peptide in vitro, displayed inhibitory activity against NDV-mediated cell fusion, while the HR121 and 5-Helix proteins, which bind the HR2 peptide in vitro, inhibited virus fusion from the avirulent NDV strain when added before the cleavage of the fusion protein. These results showed that the designed HR212, HR121 or 5-Helix protein could serve as specific antiviral agents. These data provide additional insight into the difference between the virulent and avirulent strains of NDV. 相似文献