Priyamvada Acharya

Peptides from Second Extracellular Loop of C-C Chemokine Receptor Type 5 (CCR5) Inhibit Diverse Strains of HIV-1

Background: Extracellular regions ECL2 and the N terminus of HIV coreceptor CCR5 mediate HIV-1 entry. Results: A C-terminal CCR5 ECL2 peptide inhibits HIV-1 entry and binds to gp120 of CCR5- and CXCR4-using strains. Conclusion: The binding site for CCR5 ECL2 is conserved in CCR5- and CXCR4-using viruses. Significance: Our data provide new insights into HIV-1 gp120-CCR5 interactions that can be used for inhibitor design. To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.

How an Enzyme Binds the C1 Carrier Tetrahydromethanopterin STRUCTURE OF THE TETRAHYDROMETHANOPTERIN-DEPENDENT FORMALDEHYDE-ACTIVATING ENZYME (Fae) FROM METHYLOBACTERIUM EXTORQUENS AM1

Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 Å without and at 1.9 Å with methylene-H4MPT bound. Methylene-H4MPT is bound in an “S”-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 Å and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70° that is more pronounced than that reported for free methylene-H4MPT in solution (50°). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.

Structural Biology and the Design of Effective Vaccines for HIV-1 and Other Viruses

Structural biology provides a wealth of information about the three-dimensional organization and chemical makeup of proteins. An understanding of atomic-level structure offers enormous potential to design rationally proteins that stimulate specific immune responses. Yet current vaccine development efforts makes little use of structural information. At the Vaccine Research Center, a major goal is to apply structural techniques to vaccine design for challenging pathogens, that include human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses such as influenza, Ebola, and respiratory syncytial viruses. Our three-part strategy involves 1.) the definition of the functional viral spike at the atomic level 2.) achieving a structural understanding of how neutralizing antibodies recognize the spike, and 3.) rational development of proteins that can elicit a specific antibody response. Overall, our strategy aims to incorporate information about viral spike-antibody interactions, to assimilate immunogenic feedback, and to leverage recent advances in immunofocusing and computational biology.

Conformational Changes in HIV-1 Env Trimer Induced by a Single CD4 as Revealed by Cryo-EM

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA 4. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA.

Uncleaved Soluble gp140 Constructs with Retained Native-like Trimer Conformation and Antigenicity

OA01.06 Background: Soluble gp140 constructs that mimic native-like HIV-1 Env trimers are prime immunogen candidates. gp140 cleavage into gp120 and gp41, however, is thought to be essential for mimicry of native-like conformation and antigenicity, as evidenced by binding to quaternary-specific broadly neutralizing antibodies (qbNAbs) but not to non-neutralizing antibodies (NnAbs). Methods: We employed structure-based design strategies to generate modified uncleaved soluble gp140 trimers. The designed constructs were characterized using a high-throughput ELISA assay against a panel of qbNAbs and NnAbs, as well as bio-layer interferometry, gel filtration and negative-stain EM. Results: The designed uncleaved gp140 constructs were initially tested in the context of the clade A HIV-1 strain BG505 and showed good binding to qbNAbs (including highly quaternary-specific antibodies such as PGT145 and VRC26.09) but not to NnAbs (such as F105). The constructs exhibited a significant trimer fraction by gel filtration and native-like conformation by negative-stain EM. The antigenicity and conformational properties of these constructs resembled those of the current state-of-the-art soluble gp140, BG505.SOSIP, which in contrast requires full cleavage. Conclusions: Uncleaved soluble gp140 constructs that mimic native-like trimers have the advantage of not requiring cleavage, rendering them useful candidates as protein- as well as DNA-based immunogens. Uncleaved analogs in diverse HIV-1 strains are currently being evaluated and immunogenicity studies are ongoing.