scholarly journals Molecular Determinants of Antiviral Potency of Paramyxovirus Entry Inhibitors

2007 ◽  
Vol 81 (19) ◽  
pp. 10567-10574 ◽  
Author(s):  
M. Porotto ◽  
P. Carta ◽  
Y. Deng ◽  
G. E. Kellogg ◽  
M. Whitt ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Nipah Virus ◽  
Coiled Coil ◽  
Hendra Virus ◽  
Heptad Repeat ◽  
Protein Fusion ◽  
C Peptide ◽  
Entry Inhibitors

ABSTRACT Hendra virus (HeV) and Nipah virus (NiV) constitute the Henipavirus genus of paramyxoviruses, both fatal in humans and with the potential for subversion as agents of bioterrorism. Binding of the HeV/NiV attachment protein (G) to its receptor triggers a series of conformational changes in the fusion protein (F), ultimately leading to formation of a postfusion six-helix bundle (6HB) structure and fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions, the first (the N-terminal heptad repeat [HRN]) adjacent to the fusion peptide and the second (the C-terminal heptad repeat [HRC]) immediately preceding the transmembrane domain. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing activated F molecules from forming the 6HB structure that is required for fusion. We previously reported that a human parainfluenza virus 3 (HPIV3) F peptide effectively inhibits infection mediated by the HeV glycoproteins in pseudotyped-HeV entry assays more effectively than the comparable HeV-derived peptide, and we now show that this peptide inhibits live-HeV and -NiV infection. HPIV3 F peptides were also effective in inhibiting HeV pseudotype virus entry in a new assay that mimics multicycle replication. This anti-HeV/NiV efficacy can be correlated with the greater potential of the HPIV3 C peptide to interact with the HeV F N peptide coiled-coil trimer, as evaluated by thermal unfolding experiments. Furthermore, replacement of a buried glutamic acid (glutamic acid 459) in the C peptide with valine enhances antiviral potency and stabilizes the 6HB conformation. Our results strongly suggest that conserved interhelical packing interactions in the F protein fusion core are important determinants of C peptide inhibitory activity and offer a strategy for the development of more-potent analogs of F peptide inhibitors.

scholarly journals Viral Entry Inhibitors Targeted to the Membrane Site of Action

2010 ◽  
Vol 84 (13) ◽  
pp. 6760-6768 ◽  
Author(s):  
Matteo Porotto ◽  
Christine C. Yokoyama ◽  
Laura M. Palermo ◽  
Bruce Mungall ◽  
Mohamad Aljofan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Target Cell ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Hendra Virus ◽  
Heptad Repeat ◽  
Membrane Microdomains ◽  
Site Of Action ◽  
Hiv 1

ABSTRACT The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutinin-neuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses—human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases—the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.

scholarly journals Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Beatriz Pacheco ◽  
Nirmin Alsahafi ◽  
Olfa Debbeche ◽  
Jérémie Prévost ◽  
Shilei Ding ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Envelope Glycoprotein ◽  
Coiled Coil ◽  
Bound State ◽  
Heptad Repeat ◽  
Amino Acid Residues ◽  
Entry Inhibitors ◽  
Gp41 Ectodomain ◽  
Hiv 1

ABSTRACT Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations. IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the transitions from the unliganded state to the CD4-bound state.

scholarly journals Crystal Structure and Carbohydrate Analysis of Nipah Virus Attachment Glycoprotein: a Template for Antiviral and Vaccine Design

2008 ◽  
Vol 82 (23) ◽  
pp. 11628-11636 ◽  
Author(s):  
Thomas A. Bowden ◽  
Max Crispin ◽  
David J. Harvey ◽  
A. Radu Aricescu ◽  
Jonathan M. Grimes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ebola Virus ◽  
Nipah Virus ◽  
Glycosylation Site ◽  
Hendra Virus ◽  
Molecular Surface ◽  
Spectrometric Analysis ◽  
Viral Attachment ◽  
Virus Attachment ◽  
Host Interaction

ABSTRACT Two members of the paramyxovirus family, Nipah virus (NiV) and Hendra virus (HeV), are recent additions to a growing number of agents of emergent diseases which use bats as a natural host. Identification of ephrin-B2 and ephrin-B3 as cellular receptors for these viruses has enabled the development of immunotherapeutic reagents which prevent virus attachment and subsequent fusion. Here we present the structural analysis of the protein and carbohydrate components of the unbound viral attachment glycoprotein of NiV glycoprotein (NiV-G) at a 2.2-Å resolution. Comparison with its ephrin-B2-bound form reveals that conformational changes within the envelope glycoprotein are required to achieve viral attachment. Structural differences are particularly pronounced in the 579-590 loop, a major component of the ephrin binding surface. In addition, the 236-245 loop is rather disordered in the unbound structure. We extend our structural characterization of NiV-G with mass spectrometric analysis of the carbohydrate moieties. We demonstrate that NiV-G is largely devoid of the oligomannose-type glycans that in viruses such as human immunodeficiency virus type 1 and Ebola virus influence viral tropism and the host immune response. Nevertheless, we find putative ligands for the endothelial cell lectin, LSECtin. Finally, by mapping structural conservation and glycosylation site positions from other members of the paramyxovirus family, we suggest the molecular surface involved in oligomerization. These results suggest possible pathways of virus-host interaction and strategies for the optimization of recombinant vaccines.

scholarly journals Mutations in gp120 Contribute to the Resistance of Human Immunodeficiency Virus Type 1 to Membrane-Anchored C-Peptide maC46

2009 ◽  
Vol 83 (10) ◽  
pp. 4844-4853 ◽  
Author(s):  
Felix G. Hermann ◽  
Lisa Egerer ◽  
Frances Brauer ◽  
Christian Gerum ◽  
Harald Schwalbe ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Conformational Changes ◽  
Viral Envelope ◽  
Heptad Repeat ◽  
Systematic Analysis ◽  
C Peptide ◽  
Helix Bundle ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.

scholarly journals Side Chain Packing below the Fusion Peptide Strongly Modulates Triggering of the Hendra Virus F Protein

2010 ◽  
Vol 84 (20) ◽  
pp. 10928-10932 ◽  
Author(s):  
Everett Clinton Smith ◽  
Rebecca Ellis Dutch
Keyword(s):  
Conformational Changes ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Hendra Virus ◽  
Heptad Repeat ◽  
Cell Surface Expression ◽  
Side Chain ◽  
Chain Packing ◽  
F Protein ◽  
Side Chain Packing

ABSTRACT Triggering of the Hendra virus fusion (F) protein is required to initiate the conformational changes which drive membrane fusion, but the factors which control triggering remain poorly understood. Mutation of a histidine predicted to lie near the fusion peptide to alanine greatly reduced fusion despite wild-type cell surface expression levels, while asparagine substitution resulted in a moderate restoration in fusion levels. Slowed kinetics of six-helix bundle formation, as judged by sensitivity to heptad repeat B-derived peptides, was observed for all H372 mutants. These data suggest that side chain packing beneath the fusion peptide is an important regulator of Hendra virus F triggering.

Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

2000 ◽  
Vol 20 (6) ◽  
pp. 597-612 ◽  
Author(s):  
Rebecca Ellis Dutch ◽  
Theodore S. Jardetzky ◽  
Robert A. Lamb
Keyword(s):  
Fusion Proteins ◽  
Ebola Virus ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Proteolytic Cleavage ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Hydrophobic Region ◽  
Viral Fusion Proteins

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structuralelements in the processing and function of these viral fusion proteins is discussed.

scholarly journals Spring-Loaded Heptad Repeat Residues Regulate the Expression and Activation of Paramyxovirus Fusion Protein

2007 ◽  
Vol 81 (7) ◽  
pp. 3130-3141 ◽  
Author(s):  
Laura E. Luque ◽  
Charles J. Russell
Keyword(s):  
Protein Expression ◽  
Membrane Fusion ◽  
Conformational Changes ◽  
Structural Changes ◽  
Tertiary Structure ◽  
Sendai Virus ◽  
Coiled Coil ◽  
Viral Envelope ◽  
Heptad Repeat ◽  
F Protein

ABSTRACT During viral entry, the paramyxovirus fusion (F) protein fuses the viral envelope to a cellular membrane. Similar to other class I viral fusion glycoproteins, the F protein has two heptad repeat regions (HRA and HRB) that are important in membrane fusion and can be targeted by antiviral inhibitors. Upon activation of the F protein, HRA refolds from a spring-loaded, crumpled structure into a coiled coil that inserts a hydrophobic fusion peptide into the target membrane and binds to the HRB helices to form a fusogenic hairpin. To investigate how F protein conformational changes are regulated, we mutated in the Sendai virus F protein a highly conserved 10-residue sequence in HRA that undergoes major structural changes during protein refolding. Nine of the 15 mutations studied caused significant defects in F protein expression, processing, and fusogenicity. Conversely, the remaining six mutations enhanced the fusogenicity of the F protein, most likely by helping spring the HRA coil. Two of the residues that were neither located at “a” or “d” positions in the heptad repeat nor conserved among the paramyxoviruses were key regulators of the folding and fusion activity of the F protein, showing that residues not expected to be important in coiled-coil formation may play important roles in regulating membrane fusion. Overall, the data support the hypothesis that regions in the F protein that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations regulate F protein expression and activation.

scholarly journals Inhibition of Hendra Virus Fusion

2006 ◽  
Vol 80 (19) ◽  
pp. 9837-9849 ◽  
Author(s):  
M. Porotto ◽  
L. Doctor ◽  
P. Carta ◽  
M. Fornabaio ◽  
O. Greengard ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Conformational Changes ◽  
Viral Entry ◽  
Fusion Peptide ◽  
Hendra Virus ◽  
Parainfluenza Virus ◽  
Heptad Repeat ◽  
Protein G ◽  
Human Parainfluenza Virus ◽  
Receptor Binding Protein

ABSTRACT Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions (HRN and HRC) near the fusion peptide and the transmembrane domains, respectively. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing F, after the initial triggering step, from forming the 6HB structure that is required for fusion. HeV peptides have previously been found to be effective at inhibiting HeV fusion. However, we found that a human parainfluenza virus 3 F-peptide is more effective at inhibiting HeV fusion than the comparable HeV-derived peptide.

scholarly journals Transmembrane Domain Dissociation Is Required for Hendra Virus F Protein Fusogenic Activity

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Kerri Beth Slaughter ◽  
Rebecca Ellis Dutch
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Specific Antibody ◽  
Disulfide Bonds ◽  
Transmembrane Domain ◽  
Current Model ◽  
Hendra Virus ◽  
Cell Disruption ◽  
F Protein ◽  
Fusogenic Activity

ABSTRACT Hendra virus (HeV) is a zoonotic paramyxovirus that utilizes a trimeric fusion (F) protein within its lipid bilayer to mediate membrane merger with a cell membrane for entry. Previous HeV F studies showed that transmembrane domain (TMD) interactions are important for stabilizing the prefusion conformation of the protein prior to triggering. Thus, the current model for HeV F fusion suggests that modulation of TMD interactions is critical for initiation and completion of conformational changes that drive membrane fusion. HeV F constructs (T483C/V484C, V484C/N485C, and N485C/P486C) were generated with double cysteine substitutions near the N-terminal region of the TMD to study the effect of altered flexibility in this region. Oligomeric analysis showed that the double cysteine substitutions successfully promoted intersubunit disulfide bond formation in HeV F. Subsequent fusion assays indicated that the introduction of disulfide bonds in the mutants prohibited fusion events. Further testing confirmed that T483C/V484C and V484C/N485C were expressed at the cell surface at levels that would allow for fusion. Attempts to restore fusion with a reducing agent were unsuccessful, suggesting that the introduced disulfide bonds were likely buried in the membrane. Conformational analysis showed that T483C/V484C and V484C/N485C were able to bind a prefusion conformation-specific antibody prior to cell disruption, indicating that the introduced disulfide bonds did not significantly affect protein folding. This study is the first to report that TMD dissociation is required for HeV F fusogenic activity and strengthens our model for HeV fusion. IMPORTANCE The paramyxovirus Hendra virus (HeV) causes severe respiratory illness and encephalitis in humans. To develop therapeutics for HeV and related viral infections, further studies are needed to understand the mechanisms underlying paramyxovirus fusion events. Knowledge gained in studies of the HeV fusion (F) protein may be applicable to a broad span of enveloped viruses. In this study, we demonstrate that disulfide bonds introduced between the HeV F transmembrane domains (TMDs) block fusion. Depending on the location of these disulfide bonds, HeV F can still fold properly and bind a prefusion conformation-specific antibody prior to cell disruption. These findings support our current model for HeV membrane fusion and expand our knowledge of the TMD and its role in HeV F stability and fusion promotion.

scholarly journals Mutational Analysis of Residues in the Coiled-Coil Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41

1998 ◽  
Vol 72 (12) ◽  
pp. 9676-9682 ◽  
Author(s):  
Yongkai Weng ◽  
Carol D. Weiss
Keyword(s):  
Human Immunodeficiency Virus ◽  
Glutamic Acid ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Core Structure ◽  
Heptad Repeat ◽  
Homologous Region ◽  
Fusion Activity ◽  
Wild Type ◽  
Immunodeficiency Virus

ABSTRACT The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263–273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426–430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.

Sign in / Sign up

Export Citation Format

Share Document