Structure of a V3-containing HIV-1 gp120 core.

The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is immunodominant and contains features essential for coreceptor binding. We determined the structure of V3 in the context of an HIV-1 gp120 core complexed to the CD4 receptor and to the X5 antibody at 3.5 angstrom resolution.

The SARS coronavirus S glycoprotein receptor binding domain: fine mapping and functional characterization.

The entry of the SARS coronavirus (SCV) into cells is initiated by binding of its spike envelope glycoprotein (S) to a receptor, ACE2. We and others identified the receptor-binding domain (RBD) by using S fragments of various lengths but all including the amino acid residue 318 and two other potential glycosylation sites. To further characterize the role of glycosylation and identify residues important for its function as an interacting partner of ACE2, we have cloned, expressed and characterized various soluble fragments of S containing RBD, and mutated all potential glycosylation sites and 32 other residues.

Retinoic acid-induced downmodulation of telomerase activity in human cancer cells.

Most human cancers express telomerase but its activity is highly variable and regulated by complex mechanisms. Recently, several studies have suggested that retinoic acid (RA) downregulates telomerase activity and that this effect could be a major determinant of its therapeutic activity. To elucidate possible mechanisms of RA-mediated downmodulation of telomerase activity, we measured the kinetics of concentration changes of several transcription regulators by using standard biochemical techniques at low (10 muM) and high (100 muM) RA concentrations.

Induction of prolonged survival of CD4+ T lymphocytes by intermittent IL-2 therapy in HIV-infected patients.

HIV infection leads to decreases in the number of CD4 T lymphocytes and an increased risk for opportunistic infections and neoplasms. The administration of intermittent cycles of IL-2 to HIV-infected patients can lead to profound increases (often greater than 100%) in CD4 cell number and percentage. Using in vivo labeling with 2H-glucose and BrdU, we have been able to demonstrate that, although therapy with IL-2 leads to high levels of proliferation of CD4 as well as CD8 lymphocytes, it is a remarkable preferential increase in survival of CD4 cells (with half-lives that can exceed 3 years) that is critical to the sustained expansion of these cells.

Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus.

Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus of the family Paramyxoviridae and are unique in that they exhibit a broad species tropism and cause fatal disease in both animals and humans. They infect cells through a pH-independent membrane fusion process mediated by their fusion and attachment glycoproteins. Previously, we demonstrated identical cell fusion tropisms for HeV and NiV and the protease-sensitive nature of their unknown cell receptor and identified a human cell line (HeLa-USU) that was nonpermissive for fusion and virus infection.

Human monoclonal antibodies to the S glycoprotein and related proteins as potential therapeutics for SARS.

Polyclonal antibodies have a century-old history of being effective against some viruses and, recently, monoclonal antibodies (mAbs) have also shown some clinical success. Human mAbs to the severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein have been developed by several research groups at an amazing pace. These antibodies potently neutralize infectious virus in tissue cultures and animal models, and, alone or in combination with vaccines and other drugs, may have potential for the prevention and treatment of SARS.

Marked structural and functional heterogeneity in CXCR4: separation of HIV-1 and SDF-1alpha responses.

CXCR4, the chemotactic cell receptor for SDF-1alpha, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms.

Structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor.

Angiotensin-converting enzyme 2 (ACE2) is considered an important therapeutic target for controlling cardiovascular diseases and severe acute respiratory syndrome (SARS) outbreaks. Recently solved high-resolution crystal structures of the apo-bound and inhibitor-bound forms of ACE2 have provided the basis for a novel molecular docking approach in an attempt to identify ACE2 inhibitors and compounds that block SARS coronavirus spike protein-mediated cell fusion. In this study, approximately 140 000 small molecules were screened by in silico molecular docking.

Oligomerization of the SARS-CoV S glycoprotein: dimerization of the N-terminus and trimerization of the ectodomain.

Viral envelope glycoproteins are oligomeric and the quaternary structure is critical for their membrane fusion activity. Typically the transmembrane glycoproteins of class I fusion proteins contain the oligomerization domains and the surface glycoproteins (SU) are monomeric. However, it has been previously demonstrated [J.

Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody.

The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening.

Identification of a novel CD4i human monoclonal antibody Fab that neutralizes HIV-1 primary isolates from different clades.

A new human monoclonal antibody (hmAb), designated m16, was selected by sequential antigen panning (SAP) of a human phage display library against recombinant soluble HIV-1 envelope glycoproteins (Envs) (gp140s) and their complexes with soluble CD4. It bound with high (nM) affinity to gp120 and gp140; the binding was further enhanced by interactions of the Envs with CD4. m16 inhibited cell fusion mediated by the Envs of 9 HIV-1 isolates from clades A, B, E and G with potency on average comparable to that of the broadly neutralizing human monoclonal antibody Fab X5.

Virus entry: molecular mechanisms and biomedical applications.

Viruses have evolved to enter cells from all three domains of life--Bacteria, Archaea and Eukaryotes. Of more than 3,600 known viruses, hundreds can infect human cells and most of those are associated with disease. To gain access to the cell interior, animal viruses attach to host-cell receptors.

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.

Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope.

The human monoclonal antibody Fab X5 neutralizes a broad range of HIV-1 primary isolates. The crystal structure of X5 has been determined at 1.9 A resolution.

A model of the ACE2 structure and function as a SARS-CoV receptor.

The angiotensin-converting enzyme 2 (ACE2) is an important regulator of the renin-angiotensin system and was very recently identified as a functional receptor for the SARS virus. The ACE2 sequence is similar (sequence identities 43% and 35%, and similarities 61% and 55%, respectively) to those of the testis-specific form of ACE (tACE) and the Drosophila homolog of ACE (AnCE). The high level of sequence similarity allowed us to build a robust homology model of the ACE2 structure with a root-mean-square deviation from the aligned crystal structures of tACE and AnCE less than 0.

The secret life of ACE2 as a receptor for the SARS virus.

The membrane-associated carboxypeptidase angiotensin-converting enzyme 2 (ACE2) is an essential regulator of heart function. Now, Li at al. identify and characterize an unexpected second function of ACE2 as a partner of the SARS-CoV spike glycoprotein in mediating virus entry and cell fusion.

Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library.

Identification of broadly cross-reactive human monoclonal antibodies (mAbs) has major implications for development of vaccines, inhibitors and research tools. Here we describe a sequential antigen panning (SAP) methodology that may facilitate the selection of such antibodies. An HIV-specific antibody Fab (m18) was selected from a human Fab phage-display library by SAP against several recombinant soluble HIV envelope glycoproteins (Envs) and Env-sCD4 complexes.

Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning.

Several human monoclonal antibodies can neutralize a range of human immunodeficiency virus type 1 (HIV-1) primary isolates but their potency and related ability to suppress generation of HIV-1 escape mutants is significantly lower than the activity of antiretroviral drugs currently in clinical use. Recently, a human Fab, X5, was identified and found to neutralize primary isolates from different clades. Further improvement of the potency and breadth of HIV-1 neutralization by this antibody could be critical for its potential use in the treatment of HIV-1-infected patients.

The SARS-CoV S glycoprotein: expression and functional characterization.

We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, S(e), were not cleaved to any significant degree.

A kinetic model of telomere shortening in infants and adults.

We have previously demonstrated that telomeres shorten more rapidly in peripheral mononuclear cells (PBMC) of infants than in adults (Zeichner et al., Blood 93 (1999) 2824). Here we describe a mathematical model that allows quantification of telomere dynamics both in infants and in adults.

Role of Ets/Id proteins for telomerase regulation in human cancer cells.

Most human cancers express telomerase but its activity is highly variable and regulated by complex mechanisms. Recently, we have proposed that Ets proteins may be important for regulation of telomerase activity in leukemic cells. Here we provide further evidence for the role of Ets family members and related Id proteins in telomerase regulation and characterize the underlying molecular mechanisms.

Purified complexes of HIV-1 envelope glycoproteins with CD4 and CCR5(CXCR4): production, characterization and immunogenicity.

The ability to readily elicit broadly neutralizing antibodies to HIV-1 remains elusive. We and others have hypothesized that interaction of the viral envelope glycoprotein (Env, gp120-gp41) with its receptor molecules could enhance the exposure of conserved epitopes that may facilitate the elicitation of broadly neutralizing antibodies. The Env-CD4-coreceptor complexes mediate HIV-1 entry into cells and serve as a major target for inhibitors of this process.

Evaluation of gene expression measurements from commercial microarray platforms.

Multiple commercial microarrays for measuring genome-wide gene expression levels are currently available, including oligonucleotide and cDNA, single- and two-channel formats. This study reports on the results of gene expression measurements generated from identical RNA preparations that were obtained using three commercially available microarray platforms. RNA was collected from PANC-1 cells grown in serum-rich medium and at 24 h following the removal of serum.

Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1.

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates.