Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.

To identify functional contacts between HIV-1 integrase (IN)and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys-and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The footprinting studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes. HIV-1 integrase (IN)4 is commonly viewed as an important therapeutic target for the following reasons: its catalytic activities are required for viral replication, there is no closely related cellular equivalent of IN, and specific IN inhibitors are likely to be effective against viral strains resistant to currently available therapies targeting reverse transcriptase (RT), protease, and virus-cell fusion. Detailed structural information on functional IN-DNA complexes could aid drug design efforts. For example, the promising diketo acid class of inhibitors preferentially bind to the assembled IN-viral DNA complex rather than the free protein (1-4).The two chemical reactions catalyzed by HIV-1 IN, 3Ј processing and DNA strand transfer, have been characterized in detail (reviewed in Ref. 5). First, IN removes two nucleotides from each 3Ј-end of the viral DNA synthesized by reverse transcriptase. In the following step, concerted transesterification reactions covalently join the viral DNA ends into the host genome (6). In vivo, the enzyme acts in the context of a large nucleoprotein complex with a number of viral and host proteins contributing to the integration process (7-21).HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (NTD) (residues 1-50) that contains an HHCC zinc binding motif, the catalytic core domain (CCD) (residues 51-212) containing the DDE motif essential for coordinating catalytic divalent metals, and the C-terminal domain (CTD) (residues 213-288) that is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (22-26). In addition, two-domain CCD/ CTD (27) and NTD/CCD (28) crystal structures have been determined. However,...

HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the smallmolecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN.

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here Hepatitis C virus (HCV)3 infection is a serious public health concern that affects about 170 million people worldwide (1, 2). HCV belongs to the Flaviviridae family, which comprises other human pathogens, such as dengue virus, West Nile virus, as well as yellow fever virus. The single (plus)-stranded HCV RNA genome encodes a polyprotein, which is processed into several smaller mature structural proteins, including the capsid protein (C), the envelope proteins (E1 and E2), and the nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Initial cis cleavage through NS2-NS3 releases the NS3 protein, which, in turn, continues to process the precursor (for a review,NS5B is a 65-kDa RNA-dependent RNA polymerase that is capable of initiating RNA synthesis de novo, in the absence of a primer (4 -7). However, the detailed mechanism of de novo initiation remains elusive. Crystallographic snapshots of initial stages of the reaction that include ternary or quaternary complexes of NS5B with a bound RNA template and nucleotide substrates are not yet available. Modeling studies, on the basis of structures of the apoenzyme, suggest that the enzyme must undergo extensive conformational changes to accommodate the newly synthesized double-stranded RNA (4, 8). The structure of HCV NS5B is reminiscent of a human right hand, although, in contrast to many other DNA or RNA polymerases with a similar fold, extensive interaction between the "thumb" and the tips of the "fingers" subdomains encircle the active site of NS5B (8 -10). The NTP substrates can enter through a well defined tunnel. A ␤-hairpin or flap that comprises residues Leu 443 to Ile 454 and the C-terminal tail of the protein are both located in the vicinity of the active site and may cause steric conflicts with the newly synthesized double-stranded primertemplate (4,8). At the same time, it should be noted that the precise positioning of the C terminus remains to be defined, because most of the structural and biochemical studies were performed with C-terminally truncated proteins to facilitate expression and purification procedures.HCV NS5B has also been co-crystallized with GTP (11). In addition to the active site, the authors show that GTP can bind to the thumb domain in close proximity to the fingertip ⌬1 loop that is involved in interdomain interactions between the thumb and fingers. Although this allosteric GTP site lies 30 Å away from the polymerase active site, it is implicated in the regulation of dynamic interactions between the fingers and thumb subdomains (12,13). A complex of NS5B with a short RNA oligomer (rU 5 ) provides structural information with regard to interaction between the 5Ј-end of the single-stranded template at the entrance channel of the substrate (14). The 3Ј-end of the RNA substrate is seen in the vicinity of the active site, and it is evident that binding of the short oligomer does not affect the overall fold of the enzyme. Howeve...

Achieving spin-valley coupled states is essential to promote the fantastic integration of spintronics and valleytronics. Two-dimensional transition metal systems with D3h point group, by breaking both time-reversal symmetry and structural...

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