Hyeryun Choe

The β-Chemokine Receptors CCR3 and CCR5 Facilitate Infection by Primary HIV-1 Isolates

We examined the ability of chemokine receptors and related G protein-coupled receptors to facilitate infection by primary, clinical HIV-1 isolates. CCR5, when expressed along with CD4, the HIV-1 receptor, allowed cell lines resistant to most primary HIV-1 isolates to be infected. CCR3 facilitated infection by a more restricted subset of primary viruses, and binding of the CCR3 ligand, eotaxin, inhibited infection by these isolates. Utilization of CCR3 and CCR5 on the target cell depended upon the sequence of the third variable (V3) region of the HIV-1 gp120 exterior envelope glycoprotein. The ability of various members of the chemokine receptor family to support the early stages of HIV-1 infection helps to explain viral tropism and beta-chemokine inhibition of primary HIV-1 isolates.

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.

Tyrosine Sulfation of the Amino Terminus of CCR5 Facilitates HIV-1 Entry

Chemokine receptors and related seven-transmembrane-segment (7TMS) receptors serve as coreceptors for entry of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells. Each of these otherwise diverse coreceptors contains an N-terminal region that is acidic and tyrosine rich. Here, we show that the chemokine receptor CCR5, a principal HIV-1 coreceptor, is posttranslationally modified by O-linked glycosylation and by sulfation of its N-terminal tyrosines. Sulfated tyrosines contribute to the binding of CCR5 to MIP-1 alpha, MIP-1 beta, and HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter cells expressing CCR5 and CD4. CXCR4, another important HIV-1 coreceptor, is also sulfated. Tyrosine sulfation may contribute to the natural function of many 7TMS receptors and may be a modification common to primate immunodeficiency virus coreceptors.

Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses

At least five arenaviruses cause viral haemorrhagic fevers in humans. Lassa virus, an Old World arenavirus, uses the cellular receptor α-dystroglycan to infect cells. Machupo, Guanarito, Junin and Sabia viruses are New World haemorrhagic fever viruses that do not use α-dystroglycan. Here we show a specific, high-affinity association between transferrin receptor 1 (TfR1) and the entry glycoprotein (GP) of Machupo virus. Expression of human TfR1, but not human transferrin receptor 2, in hamster cell lines markedly enhanced the infection of viruses pseudotyped with the GP of Machupo, Guanarito and Junin viruses, but not with those of Lassa or lymphocytic choriomeningitis viruses. An anti-TfR1 antibody efficiently inhibited the replication of Machupo, Guanarito, Junin and Sabia viruses, but not that of Lassa virus. Iron depletion of culture medium enhanced, and iron supplementation decreased, the efficiency of infection by Junin and Machupo but not Lassa pseudoviruses. These data indicate that TfR1 is a cellular receptor for New World haemorrhagic fever arenaviruses.

Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP1,2) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.

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.

HIV-1 ENTRY AND MACROPHAGE INFLAMMATORY PROTEIN-1BETA -MEDIATED SIGNALING ARE INDEPENDENT FUNCTIONS OF THE CHEMOKINE RECEPTOR CCR5

The human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to CD4 to enter its target cell. The chemokine receptor CCR5 is used by macrophage-tropic strains of HIV-1, which predominate during the asymptomatic stages of infection. Here we investigate whether the ability of CCR5 to signal in response to its β-chemokine ligands is necessary or sufficient for viral entry. Three CCR5 mutants with little or no ability to mobilize calcium in response to macrophage inflammatory protein-1β could nonetheless support HIV-1 entry and the early steps in the virus life cycle with efficiencies comparable with those of wild-type CCR5. Conversely, a chimeric receptor with the N terminus of CCR2 replacing that of CCR5 responded to macrophage inflammatory protein-1β and MCP-1 but did not efficiently support viral entry. These results demonstrate that chemokine signaling and HIV-1 entry are separable functions of CCR5 and that only viral entry requires the N-terminal domain of CCR5.

The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1α association and HIV-1 entry

The chemokine receptor CXCR4 plays critical roles in development, immune function, and human immunodeficiency virus type 1 (HIV-1) entry. Here we demonstrate that, like the CC-chemokine receptors CCR5 and CCR2b, CXCR4 is posttranslationally modified by sulfation of its amino-terminal tyrosines. The sulfate group at tyrosine 21 contributes substantially to the ability of CXCR4 to bind its ligand, stromal derived factor 1α. Tyrosine sulfation plays a less significant role in CXCR4-dependent HIV-1 entry than in CCR5-dependent HIV-1 entry. In some cell lines, CXCR4 is efficiently modified by a chondroitin sulfate chain at serine 18, but neither HIV-1 entry nor stromal derived factor 1α binding was affected by loss of this glycosaminoglycan. These data demonstrate a functional role for tyrosine sulfate in the CXC-chemokine receptor family and underscore a general difference in HIV-1 utilization of CCR5 and CXCR4.

Tyrosine Sulfation of Human Antibodies Contributes to Recognition of the CCR5 Binding Region of HIV-1 gp120

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.

SARS Coronavirus, but Not Human Coronavirus NL63, Utilizes Cathepsin L to Infect ACE2-expressing Cells

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.