Biomedical subjects

Michael Farzan

Publications and source records attributed to Michael Farzan.

At least 37 records · Page 2Linked to original sources

PubMed2005

An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor.

The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

Cell Line↗

PubMed2005

Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2.

Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002-2003 SARS outbreak and during the much less severe 2003-2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002-2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002-2003 SARS epidemic.

Amino Acid Sequence↗

PubMed2005

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC).

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.

Animals↗

PubMed2005

Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion.

Summary Plasmodium vivax depends on interaction with the Duffy antigen/receptor for chemokines (DARC) for invasion of human erythrocytes. The 140 kDa P. vivax Duffy-binding protein (PvDBP) mediates interaction with DARC. The receptor-binding domain of PvDBP maps to its N-terminal, cysteine-rich region, region II (PvRII), which contains approximately 300 amino acid residues including 12 conserved cysteines. Using surface plasmon resonance, we show that binding of PvRII to DARC is a high-affinity interaction with a binding constant (K(D)) of 8.7 nM. The minimal binding domain of PvRII has been previously mapped to a central 170-amino-acid stretch that includes cysteines 5-8. Here, we have used site-directed mutagenesis and quantitative binding assays to map amino acid residues within PvRII that make contact with DARC. Of the seven alanine replacement mutations that had an effect on binding, five were mutations in hydrophobic residues suggesting that hydrophobic interactions play a major role in the interaction of PvDBP with DARC. Genetic diversity studies have shown that six of the seven binding residues identified in PvRII are conserved in P. vivax field isolates, which provides support for their role in interaction with DARC.

Animals↗

PubMed2005

Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants.

In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.

Amino Acid Substitution↗

PubMed2005

Functional mimicry of a human immunodeficiency virus type 1 coreceptor by a neutralizing monoclonal antibody.

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 beta19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 loop base determined 412d preference for CCR5-using HIV-1 strains. A chimeric molecule in which the 412d heavy-chain third complementarity-determining loop sequence replaces the CCR5 N terminus functioned as an efficient second receptor, selectively supporting the entry of CCR5-using HIV-1 strains. Sulfation of N-terminal tyrosines contributed to the function of this chimeric receptor. These results emphasize the close mimicry of the CCR5 N terminus by the gp120-interactive region of a naturally elicited CD4i antibody.

Amino Acid Sequence↗

PubMed2005

ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia.

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

Angiotensin-Converting Enzyme 2↗

PubMed2005

Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region.

Immunization with a killed or inactivated viral vaccine provides significant protection in animals against challenge with certain corresponding pathogenic coronaviruses (CoVs). However, the promise of this approach in humans is hampered by serious concerns over the risk of leaking live severe acute respiratory syndrome (SARS) viruses. In this study, we generated a SARS vaccine candidate by using the live-attenuated modified vaccinia virus Ankara (MVA) as a vector. The full-length SARS-CoV envelope Spike (S) glycoprotein gene was introduced into the deletion III region of the MVA genome. The newly generated recombinant MVA, ADS-MVA, is replication incompetent in mammalian cells and highly immunogenic in terms of inducing potent neutralizing antibodies in mice, rabbits, and monkeys. After two intramuscular vaccinations with ADS-MVA alone, the 50% inhibitory concentration in serum was achieved with reciprocal sera dilutions of more than 1,000- to 10,000-fold in these animals. Using fragmented S genes as immunogens, we also mapped a neutralizing epitope in the region of N-terminal 400 to 600 amino acids of the S glycoprotein (S400-600), which overlaps with the angiotensin-converting enzyme 2 (ACE2) receptor-binding region (RBR; S318-510). Moreover, using a recombinant soluble RBR-Fc protein, we were able to absorb and remove the majority of the neutralizing antibodies despite observing that the full S protein tends to induce a broader spectrum of neutralizing activities in comparison with fragmented S proteins. Our data suggest that a major mechanism for neutralizing SARS-CoV likely occurs through blocking the interaction between virus and the cellular receptor ACE2. In addition, ADS-MVA induced potent immune responses which very likely protected Chinese rhesus monkeys from pathogenic SARS-CoV challenge.

Angiotensin-Converting Enzyme 2↗

PubMed2005

JLK inhibitors: isocoumarin compounds as putative probes to selectively target the gamma-secretase pathway.

Alzheimer's disease is characterized by the extracellular deposition of the amyloid beta-peptide that derives from its precursor betaAPP by sequential actions of beta- and gamma- secretases, respectively. Recent studies aimed at identifying these enzymes have been reported as it is thougth that their inhibition should hopefully lead to reduce Abeta load in the AD brains. beta-secretase seems to be due to BACE1, a novel membrane-bound aspartyl protease. gamma-secretase identification is still a matter of controversy. Invalidation of presenilin genes was reported to impair both gamma-secretase-mediated Abeta production and Notch cleavage leading to NICD production. This observation together with another biochemical and pharmacological evidences led to suggest that presenilins could be the genuine long-searched gamma-secretase that would be responsible for both APP and Notch cleavages. We have designed novel non peptidic potential inhibitors of gamma-secretase (referred to as JLK inhibitors) and examined their ability to prevent Abeta40 and Abeta42 secretions as well as NICD production. Three out of a series of these agents drastically lower the recoveries of both Abeta40 and Abeta42 produced by betaAPP-expressing cell lines and concomitantly protect intracellular C99 and C83 recoveries. These inhibitors also prevent Abeta40/42 productions by C99-expressing cells. Interestingly, these inhibitors were totally unable to affect the DeltaENotch cleavage leading to NICD generation. Here, we also further characterize the pharmacological properties and specificity of these JLK inhibitors.

Amyloid Precursor Protein Secretases↗

PubMed2004

Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine.

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS.

Animals↗

PubMed2004

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins.

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.

Asparagine↗

PubMed2004

Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association.

Effective prophylaxis and antiviral therapies are urgently needed in the event of reemergence of the highly contagious and often fatal severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) infection. We have identified eight recombinant human single-chain variable region fragments (scFvs) against the S1 domain of spike (S) protein of the SARS-CoV from two nonimmune human antibody libraries. One scFv 80R efficiently neutralized SARS-CoV and inhibited syncytia formation between cells expressing the S protein and those expressing the SARS-CoV receptor angiotensin-converting enzyme 2 (ACE2). Mapping of the 80R epitope showed it is located within the N-terminal 261-672 amino acids of S protein and is not glycosylation-dependent. 80R scFv competed with soluble ACE2 for association with the S1 domain and bound S1 with high affinity (equilibrium dissociation constant, Kd=32.3 nM). A human IgG1 form of 80R bound S1 with a 20-fold higher affinity of 1.59 nM comparable to that of ACE2 (Kd=1.70 nM), and neutralized virus 20-fold more efficiently than the 80R scFv. These data suggest that the 80R human monoclonal antibody may be a useful viral entry inhibitor for the emergency prophylaxis and treatment of SARS, and that the ACE2-binding site of S1 could be an attractive target for subunit vaccine and drug development.

Amino Acid Sequence↗

PubMed2004

Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120.

The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective V(H)-gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.

Acquired Immunodeficiency Syndrome↗

PubMed2004

CD4-induced T-20 binding to human immunodeficiency virus type 1 gp120 blocks interaction with the CXCR4 coreceptor.

The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.

Amino Acid Sequence↗

PubMed2004

Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2.

Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2). Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-protein-pseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.

Amino Acid Sequence↗

PubMed2004

Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2.

Replication of viruses in species other than their natural hosts is frequently limited by entry and postentry barriers. The coronavirus that causes severe acute respiratory syndrome (SARS-CoV) utilizes the receptor angiotensin-converting enzyme 2 (ACE2) to infect cells. Here we compare human, mouse, and rat ACE2 molecules for their ability to serve as receptors for SARS-CoV. We found that, compared to human ACE2, murine ACE2 less efficiently bound the S1 domain of SARS-CoV and supported less-efficient S protein-mediated infection. Rat ACE2 was even less efficient, at near background levels for both activities. Murine 3T3 cells expressing human ACE2 supported SARS-CoV replication, whereas replication was less than 10% as efficient in the same cells expressing murine ACE2. These data imply that a mouse transgenically expressing human ACE2 may be a useful animal model of SARS.

3T3 Cells↗

PubMed2003

A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2.

The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC(50) of less than 10 nm, whereas the IC(50) of the S1 domain was approximately 50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.

Alanine↗

PubMed2003

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.

Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.

Angiotensin-Converting Enzyme 2↗