Search

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis.sup.1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance.sup.6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment--which contributes to virologic failure, resistance generation and viral transmission--as well as of pre-exposure prophylaxis, leading to new infections.sup.1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence.sup.10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log.sub.10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV. The small molecule GS-6207, which disrupts the function of the HIV capsid protein, shows potential as a long-acting therapeutic agent for the treatment and prevention of HIV infection., Author(s): John O. Link [sup.1] , Martin S. Rhee [sup.1] , Winston C. Tse [sup.1] [sup.8] , Jim Zheng [sup.1] , John R. Somoza [sup.1] , William Rowe [sup.1] , [...]

Broadly reactive antibodies targeting the influenza A virus hemagglutinin (HA) head domain are thought to be rare and to require extensive somatic mutations or unusual structural features to achieve breadth against divergent HA subtypes. Here we describe common genetic and structural features of protective human antibodies from several individuals recognizing the trimer interface (TI) of the influenza A HA head, a recently identified site of vulnerability. We examined the sequence of Tl-reactive antibodies, determined crystal structures for TI antibody-antigen complexes, and analyzed the contact residues of the antibodies on HA to discover common genetic and structural features of TI antibodies. Our data reveal that many TI antibodies are encoded by a light chain variable gene segment incorporating a shared somatic mutation. In addition, these antibodies have a shared acidic residue in the heavy chain despite originating from diverse heavy chain variable gene segments. These studies show that the TI region of influenza A HA is a major antigenic site with conserved structural features that are recognized by a common human B cell public clonotype. The canonical nature of this antibody-antigen interaction suggests that the TI epitope might serve as an important target for structure-based vaccine design., Introduction Influenza A virus (IAV) is one of the most common causes of severe lower respiratory illness in humans and exhibits a wide antigenic diversity in circulating field strains. Seasonal [...]

Author(s): Robert N. Kirchdoerfer [1]; Christopher A. Cottrell [1]; Nianshuang Wang [2]; Jesper Pallesen [1]; Hadi M. Yassine [3]; Hannah L. Turner [1]; Kizzmekia S. Corbett [3]; Barney S. Graham [...]

Residues within processed protein fragments bound to major histocompatibility complex class I (MHC-I) glycoproteins have been considered to function as a series of 'independent pegs' that either anchor the peptide (p) to the MHC-I and/or interact with the spectrum of [alpha][beta]-T-cell receptors (TCRs) specific for the pMHC-I epitope in question. Mining of the extensive pMHC-I structural database established that many selfand viral peptides show extensive and direct interresidue interactions, an unexpected finding that has led us to the idea of 'constrained' peptides. Mutational analysis of two constrained peptides (the HLA B44 restricted self-peptide (B44DP[alpha]-EEFGRAFSF) and an H2-[D.sup.b] restricted influenza peptide (DbPA, SSLENFRAYV) demonstrated that the conformation of the prominently exposed arginine in both peptides was governed by interactions with MHC-I-orientated flanking residues from the peptide itself. Using reverse genetics in a murine influenza model, we revealed that mutation of an MHC-I-orientated residue (SSLENFRAYVSSLENARAYV) within the constrained PA peptide resulted in a diminished cytotoxic T lymphocyte (CTL) response and the recruitment of a limited pMHC-I specific TCR repertoire. Interactions between individual peptide positions can thus impose fine control on the conformation of pMHC-I epitopes, whereas the perturbation of such constraints can lead to a previously unappreciated mechanism of viral escape. crystal structure | cytotoxic T cell | MHC class I | viral immunity | epitope www.pnas.org/cgi/doi/10.1073/pnas.1000032107

The M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Structure determination by solid-state and solution NMR and X-ray crystallography has contributed significantly to our understanding, but channel activation may involve conformations not captured by these studies. Indeed, solid-state NMR data demonstrate that the M2 protein possesses significant conformational heterogeneity. Here, we report molecular dynamics (MD) simulations of the M2 transmembrane domain (TMD) in the absence and presence of the antiviral drug amantadine. The ensembles of MD conformations for both apo and bound forms reproduced the NMR data well. The TMD helix was found to kink around Gly-34, where water molecules penetrated deeply into the backbone. The amantadine-bound form exhibited a single peak [approximately equal to] 10[degrees] in the distribution of helix-kink angle, but the apo form exhibited 2 peaks, [approximately equal to] 0[degrees] and 40[degrees]. Conformations of the apo form with small and large kink angles had narrow and wide pores, respectively, around the primary gate formed by His-37 and Trp-41. We propose a structural model for channel activation, in which the small-kink conformations dominate before proton uptake by His-37 from the exterior, and proton uptake makes the large-kink conformations more favorable, thereby priming His-37 for proton release to the interior. solid-state NMR | MD simulations | helix kink | histidine tetrad | amantadine

The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to [approximately equal to]17-[Angstrom] resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes. cryoelectron tomography | virus assembly | contrast transfer function | capsid | retrovirus

The kernel energy method (KEM) is applied to the vesicular stomatitis virus (VSV) nucleoprotein (PDB ID code 2QVJ). The calculations employ atomic coordinates from the crystal structure at 2.8-[Angstrom] resolution, except for the hydrogen atoms, whose positions were modeled by using the computer program HYPERCHEM. The calculated KEM ab initio limited basis Hartree-Fock energy for the full 33,175 atom molecule (including hydrogen atoms) is obtained. In the KEM, a full biological molecule is represented by smaller 'kernels' of atoms, greatly simplifying the calculations. Collections of kernels are well suited for parallel computation. VSV consists of five similar chains, and we obtain the energy of each chain. Interchain hydrogen bonds contribute to the interaction energy between the chains. These hydrogen bond energies are calculated in Hartree-Fock (HF) and Moller-Plesset perturbation theory to second order (MP2) approximations by using 6-31G** basis orbitals. The correlation energy, included in MP2, is a significant factor in the interchain hydrogen bond energies. Hartree-Fock | KEM | Moller-Plesset | quantum mechanics

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-[beta] mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3' end processing of all cellular pre-mRNAs. Here we report the 1.95-[Angstrom] resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-[beta] pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex. antiviral drug discovery | bird flu | vaccine engineering | virology | X-ray crystallography

Author(s): Gabriel L. Butterfield [1, 2, 3]; Marc J. Lajoie [1, 2]; Heather H. Gustafson [4, 5]; Drew L. Sellers [4, 5, 6]; Una Nattermann [1, 2, 7]; Daniel Ellis [...]

A study was conducted to examine the rotational dynamics of the M2 transmembrane peptide (M2TMP) helical bundle under different sample preparation conditions and in lipid bilayers of different thickness. The result data demonstrate that the tilt angle of the M2TMP helices depends on the membrane thickness to reduce the hydrophobic mismatch.

Hepatitis C virus (HCV) is a major cause of chronic liver disease that frequently leads to steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCV core protein is not only a component of viral particles but also a multifunctional protein because liver steatosis and HCC are developed in HCV core gene-transgenic (CoreTg) mice. Proteasome activator PA28[gamma],/REG[gamma] regulates host and viral proteins such as nuclear hormone receptors and HCV core protein. Here we show that a knockout of the PA28[gamma] gene induces the accumulation of HCV core protein in the nucleus of hepatocytes of CoreTg mice and disrupts development of both hepatic steatosis and HCC. Furthermore, the genes related to fatty acid biosynthesis and srebp-1c promoter activity were up-regulated by HCV core protein in the cell line and the mouse liver in a PA28[gamma]-dependent manner. Heterodimer composed of liver X receptor [alpha] (LXR[gamma]) and retinoid X receptor [alpha] (RXR[alpha]) is known to up-regulate srebp-1c promoter activity. Our data also show that HCV core protein enhances the binding of LXR[alpha]/RXR[alpha] to LXR-response element in the presence but not the absence of PA28[gamma]. These findings suggest that PA28[gamma] plays a crucial role in the development of liver pathology induced by HCV infection. fatty acid | proteasome | sterol regulatory element-binding protein (SREBP) | RXR[alpha] | LXR[alpha]

Time-resolved and steady-state fluorescence of the intrinsic protein fluorophore tryptophan is used to study the bacteriophage T4 capsid protein gp23. Denaturation of the N-terminus in hexameric wild type gp23 is observed around 40 degrees C, additionally the lifetimes obtained from time-resolved fluorescence measurements could tentatively be assigned to specific trytophan residues with the help of homology model of gp23.

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription/ export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA. mRNA export | nucleolus | virus

Influenza A virus NS1 is a multifunctional protein, and in virusinfected cells NS1 modulates a number of host-cell processes by interacting with cellular factors. Here, we report that NS1 binds directly to p85[beta], a regulatory subunit of phosphatidylinositol-3-kinase (PI3K), but not to the related p85[alpha] subunit. Activation of PI3K in influenza virus-infected cells depended on genome replication, and showed kinetics that correlated with NS1 expression. Additionally, it was found that expression of NS1 alone was sufficient to constitutively activate PI3K, causing the phosphorylation of a downstream mediator of PI3K signal transduction, Akt. Mutational analysis of a potential SH2-binding motif within NS1 indicated that the highly conserved tyrosine at residue 89 is important for both the interaction with p85[beta], and the activation of PI3K. A mutant influenza virus (A/Udorn/72) expressing NS1 with the Y89F amino acid substitution exhibited a small-plaque phenotype, and grew more slowly in tissue culture than VVT virus. These data suggest that activation of PI3K signaling in influenza A virus-infected cells is important for efficient virus replication. Akt phosphorylation | multifunctional NS1 protein | reverse genetics

In recent years, structural studies have identified a number of bacterial, viral, and eukaryotic adhesive proteins that have a trimeric architecture. The prototype examples in bacteria are the Haemophilus influenzae Hia adhesin and the Yersinia enterocolitica YadA adhesin. Both Hia and YadA are members of the trimericautotransporter subfamily and are characterized by an internal passenger domain that harbors adhesive activity and a short C-terminal translocator domain that inserts into the outer membrane and facilitates delivery of the passenger domain to the bacterial surface. In this study, we examined the relationship between trimerization of the Hia and YadA passenger domains and the capacity for adhesive activity. We found that subunit-subunit interactions and stable trimerization are essential for native folding and stability and ultimately for full-level adhesive activity. These results raise the possibility that disruption of the trimeric architecture of trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity.

High-resolution structures of the influenza A/M2 protein transmembrane domain at high and low pH were compared to understand the pH-dependent conformational changes which might facilitate proton conduction by alternately changing the accessibility of the N-terminal and C-terminal regions of the channel as a proton transits through the transmembrane domain. The results demonstrated that M2 functionally reconstituted in liposomes populates at least three different conformational states over a physiologically relevant pH range, with transition midpoints that are consistent with previously reported His37 p[K.sub.a] values.

The self-assembly properties of highly purified, recombinant L4-22K protein is characterized to understand the thermodynamics of assembly of two human adenoviral (Ad) proteins, IVa2 and L4-22K. L4-22K which reversibly assembles into higher-order structures according to an indefinite, isodesmic assembly scheme could provide significant insight into the thermodynamics of assembly of L4-22K and IVa2 on the PS.

Several in vivo, in vitro and structural studies are conducted to explain the mechanisms involved in the myristate exposure taking place in the human immunodeficiency virus type 1 matrix protein. The analyzed exposure is shown to be highly modulated by the pH.

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, are prepared to construct a glycan array and are used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes. The glycan array is used to profile the binding pattern of different influenza viruses, the binding patterns of viruses and HA proteins are similar and the glycosylation at Asn27 is necessary for receptor binding.

Several mass spectrometry techniques are employed to explain the structure of the outer domain exhibited by the glycosylated and unliganded gp 120-based antigen. The interactions taking place between the specific sugar moieties and amino acid side chains are shown to be the main determinants of the observed structure.

DNA amphiphile particles template formation of virus capsids is reported. A general method for loading virus nanocarriers is also reported.

Infrared spectroscopy is used for monitoring structural changes occurring in hepatitis C virus (HCV) core protein and loop IIId upon formation of nucleocapsid-like particles (NLPs). Intermolecular protein-nucleic acid contacts in the particles have involved RNA phosphate groups and positively charged amino acid residues such as arginine and lysine.

The function, structure and mechanism of the vacuolar ([H.sup.+])-ATPases are described. The role of V-ATPase subunit isoforms and the mechanisms involved in regulation of V-ATPase activity is discussed.

Xenon sensor molecules are incorporated in the interior of an MS2 viral capsid, conferring multivalency and other properties of the viral capsid to the sensor molecule. The resulting signal amplification has facilitated the detection of sensor and the amplification has promised the detection of chemical targets at much lower concentrations than would be possible without the capsid scaffold.

A study was conducted to demonstrate the effect of amino acid polymorphisms on the structural stability and domain cooperativity of gp120 and the differencec reflected in the binding mechanism of the viral envelope glycoprotein to the cell surface receptor and coreceptor. The results for the effects of gp120 polymorphisms on binding to host cell receptors highlights the rules for the future development of entry inhibitors that must recognize and deal with genomic differences between HIV strains.

Coiled-coil arrays were used to study the interaction specificity of four viral basic-region leucine-zipper transcription factors (bZIPs) and assess their self-associations as well as heterointeractions with 33 representative human bZIPs. The new interactions detected for HBZ with MAFB, MAFG, ATF2, CEBPG, and CREBZF and MEQ with NFIL3 were confirmed in solution by circular dichroism.

A solid-state NMR analysis of the wild-type HIV-1 CA protein, prepared as conical and spherical assemblies that are stable and are not affected by magic angle spinning of the samples, is presented. The results have shown that the assemblies of CA protein are amenable to the structural analysis by solid-state NMR spectroscopy.

The interaction of the human papillomavirus E7 oncoprotein (HPV16 E7) with the DNA binding domain of the E2 protein (E2C) which leads to ionic strength-dependent hetero-oligomerization even at the lowest measurable concentrations is described. Sequestering E2 into E7-E2 oligomers is found to provide a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome.

The application of the various heterodimeric coiled-coil peptide oligomers for the controlled encapsulation of the multiple proteins in virus capsids is demonstrated. The genetic modification mechanism is shown to be extremely effective for a controlled and efficient encapsulation.

A different mode of human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC)/nucleic acid (NA) interaction, including the NC-induced sharp bending of short segments of fully duplexed DNA/DNA and DNA/RNA, is described. Single-molecule fluorescence resonance energy transfer (SM-FRET) is used for probing NC-induced NA bending and associated heterogeneous conformational dynamics for model NC/NA complexes.

The static and dynamic laser light-scattering methods are used for studying the kinetic properties of the self-assembly of hydrophilic Keplerate-type polyoxometalate (POM) {[Mo.sub.77][Fe.sub.30]} macroanions into single-layer, vesicle-like blackberry structures in solutions. The kinetics of the blackberry formation of the {[Mo.sub.77][Fe.sub.30]} macroanions have shown similarities of the self-assembly of virus capsid proteins into spherical capsid shells, which has shown possible connections between the self-assembly behaviors of inorganic species and biological macromolecules.

Purification and characterization of recombinant full-length gpV and its C-terminal domain found in P2 phage virion which have tail spike proteins beneath the baseplate to adsorb to the outer membrane of Escherichia coli during the infection process is reported. The results concluded that the C-terminal domain of gpV is a stable trimer and serves as the receptor-binding domain for the second step in the phage adsorption process.

An efficient oxidative coupling method which involves the periodate-mediated reaction of phenylene diamine substituted oligonucleotides with aniline groups installed on the outer surface of the capsid shells was developed to attach nucleic acid aptamers to the surface of a genome-free viral capsid carrier. The study observations suggested that aptamer-labeled capsids could be used for the targeted drug delivery of acid-labile prodrugs that would be preferentially released upon lysosomal acidification.

Steady-state and time-resolved fluorescence experimental studies were conducted to understand the binding kinetics of nucleocapsid protein (NC) of HIV-1 which is a highly conserved protein essential for the virus life cycle that constitutes an attractive target for new antiviral agents. The results indicated that NC binding strongly increased the fluorescence quantum yield of 2-aminopurine (2AP) by reducing the dynamic quenching of 2Ap by its close neighbors and slowing the picosecond to nanosecond conformational fluctuations of the oligonucleotides.

The structural characterization of N-terminal RNA-binding domain of protein B2 from nodamura virus (NMV B2), a member of the nodavirus family which acts as a suppressor of RNA interference (RNAi) is described. A dimeric, helical bundle structure packing of NMW B2 that placed the RNA-binding residues along one face of symmetry-related molecules suggested a potential application for recognition of dsRNA.

The various features of the assembly inhibitors formed by the combination of the assembly domain of the hepatitis B virus capsid protein and the defective mutant, Cp149-Y132A are discussed. These coassembled capsids are shown to be extremely fragile, as compared to the conventional ones.

Molecular simulations have provided a means of analyzing self-assembly of viral proteins into icosahedral capsids. The studies have shown the complex processes that govern viral capsid assembly and have suggested some features of the assembly process that can be used for controlling the assembly of icosahedral capsids and nonicosahedral capsules.

A study is conducted to determine if binding of the intrinsically disordered T cell receptor (TCR) cytoplasmic tail to the well-folded simian immunodeficiency virus Nef core domain is accompanied by a disorder-to-order transition. The 1:1 complex formed by TCR with Nef that remains unfolded in the complex has suggested existence of specific interactions for unfolded protein molecules.

The nucleic acid binding properties of the Cucumovirus RNAi suppressor tomato aspermy virus protein 2B (TAV 2B) are studied by using total internal reflection fluorescence spectroscopy (TIRFS). Binding to RNA has oligomerized and induced a conformational change in TAV 2B, causing it to form a primarily helical structure and a 4:2 protein-RNA complex.

A yellow fluorescent protein-MreB conjugate in merodiploid strain SC9 is used to study the behavior of MreB in Vibrio parahaemolyticus under various stresses, which could have significant implications for study of the cellular stress response and aging. The expression of MreB is declined in bacteria in the stationary phase and under starvation but is upregulated during the initial periods of cold shock and viable but nonculturable (VBNC) state differentiation.

A study was conducted to characterize the structural and dynamic properties of S2-HR2 which is one of the heptad repeat domains of the envelope glycoprotein from severe acute respiratory syndrome coronavirus (SARS-CoV). The results obtained for the transition state pathway of S2-HR2 from the prefusion state to the postfusion state might provide insights into the design of structure-based antagonists of SARS-CoV as well as other enveloped viruses.

A detailed high-resolution characterization of the membrane proximal external region (MPER) was performed to explore the structure-function relationship of glycoprotein (gp41) and its role in the HIV-1 fusion. The results obtained revealed that MPER stabilize the six-helix bundle (6-HB) and are participate in the interaction of T-20 (FUZEON, an anti-HIV-1 fusion inhibitor drug) with the gp41 fusion peptide proximal region (gp41 FP-PR).

The study presents a structural and dynamic characterization of the interaction that takes place between the putative fusion peptide of the S2 SARS coronavirus (SARS-CoV) virus protein and the lipid membranes. The putative fusion domain of SARS-CoV is shown to lead to the merging of the viral and target cell membranes.

The NMR structure of the W184A/M185A C-terminal domain (CTD) in its monomeric form is described. The studies have shown that dimerization-induced conformational changes are present in the two crystal structures of the CTD dimers and have suggested a mechanism that can accommodate both of the different dimer models playing functional roles during the Gag assembly of the immature capsids.

The stoichiometry of the newly developed NS3-NS5B oligomeric protein complex which could be used for treatment of liver diseases caused by Hepatitis C virus (HCV) is described.

Structural analysis of domain 2 of HCV NS5A (nonstructural protein 5A protein) (NS5A-D2) is conducted using multinuclear magnetic resonance spectroscopy. The results show that NS5A-D2 exists intrinsically unfolded but it still maintains in its functional state.