eradicated as HIV-‐1 has rarely been totally cleared from an individual 55,56 and a vaccine is still missing.
The reasons why the search for an effective vaccine has been unsuccessful until now could be in part the inability of the immune system to detect a dormant virus and inherent to the tropism of the virus that targets to destruction the immune cells themselves 44. Another important point that could explain the failure of the immune system to detect HIV-‐1 and mount a robust response is that this virus does not productively infect the DCs that are antigen-‐presenting cells (APCs), that prime the immune effectors to kill infected cells. Yet the antigen from these cells is being presented. It seems likely that the APC needs to be activated for the priming of effector T cells to be efficient, an unproductive infection leading to no immune activation will fail to fulfill this prerequisite 44.
The innate immunity actors and consequences of their activation will be introduced further below.
1.1.7 The structures of the HIV-‐1 virion and genome
The HIV-‐1 gag orf codes for a precursor polyprotein of 55 kDa in size, called Pr55gag, which is cleaved within the virion into the MA, the CA, the NC proteins and p6 that is involved in viral budding 57.
HIV-‐1 membrane form a spherical particle that has a diameter of approximately 110 nanometers (figure 4). The virion contains a conical-‐shaped CA protein complex that is composed of 216 hexamers and 12 pentamers, linked between them by the C-‐terminal domains of CA 58,59(figure 4).
The viral particle core is enclosed by a layer of MA proteins, in turn surrounded by a lipid bilayer coming from previous infection events.
The MA protein form hexameric higher-‐order complexes, which encapsulate the viral core. These complexes interact with different virion components and seem
to be essential for various processes. The well demonstrated bindings include the interaction with envelope bilayer through myristoylated motifs, a process essential for virion assembly at the plasma membrane 57. The MA interacts as well with phosphatidiylinositol-‐4,5-‐bisphosphate, leading to the targeting of the myristoyl tails to the plasma membrane and helping the MA to bind to the viral genome 60,61. Although subject to controversy, MA was reported to bind to the inner domain of gp41, stabilizing the interaction of the envelope into the assembling virion 57. Additionally, the Pr55gag-‐derived protein was shown to interact with the reverse-‐transcription and pre-‐integration complexes (RTC and PIC, respectively), suggesting a role of the MA complex in the early viral life-‐cycle steps.
The CA protein is divided into two structural domains. The N-‐terminal domain stabilizes the structure of the virion and is in the outer layer of the viral core. The C-‐terminal domain faces the inner space containing the genome and contributes to link the hexameric and pentameric rings together. The hexameric lattice of CA interacts with different cellular factors. Its binding to the restriction factor TRIM5 inhibits both reverse-‐transcription and further pre-‐integration steps (see below). As it will be further discussed later, the CA interacts with endogenous Cyclophilin A.
When Cyclophilin A is part of a TRIM5 orthologue protein, HIV-‐1 is bound and strongly restricted 62. The restriction mediated by the TRIM5 protein and orthologues will be discussed in more detail in the next sections.
incorporation of pentamers in the mature capsid, which in turn dictates the lattice curvature. Despite limited evidence from previous solution NMR studies that the N- and C-terminal domains do not appear to interact with one another in the full-length capsid protein,17the overall magnitude and time scale of the relative motions between the domains are unknown, and the conformational space sampled by the domains has not yet been characterized.
Here we explore the conformational space sampled by the monomeric and dimeric species of the wild-type, full-length HIV-1 capsid protein, CAFL (Figure 2), using experimental NMR residual dipolar couplings (RDCs) and small- and wide-angle solution X-ray scattering (SAXS/WAXS) data in an
To date, attempts to study the full-length capsid protein by conventional solution NMR methods have been hampered by severe resonance line-broadening of the backbone resonances of the linker residues, as well as of residues at the dimer interface as a consequence of a dynamic monomer/dimer exchange. Although such localized line broadening is an impediment for traditional NMR structure determination, it can be circumvented, providing that a limited number of RDCs can be measured within each domain of the full-length capsid protein and the structures of the individual domains in the full-length capsid and the isolated domain constructs are the same.
Under these conditions, the individual domains of a multi-domain protein/macromolecular assembly can be treated as rigid bodies for ensemble simulated annealing calculations in which RDCs arising from steric alignment provide both shape and orientational information,18,19while the SAXS/WAXS data provide complementary restraints on size and shape.20,21This hybrid approach is much less time-consuming than conven-tional methods of NMR structure determination20and can be readily transferred to other multidomain proteins.
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MATERIALS AND METHODSProtein Expression and Purification. All full-length HIV-1 capsid constructs, the wild-type (CAFL, residues 1−231, plasmid pNL4-3), the disulfide-linked mutant (CAFLV181C), and the monomeric mutant (CAFLW184/M185A), as well as the four C-terminal domain constructs, CA144−231and CA146−231and the corresponding disulfi de-linked-linked mutants (CA144−231V181C and CA146−231V181C ) (see Figure 2), were subcloned in a pET-11a vector and expressed in BL21-CodonPlus (DE3)-RIPL competent cells (Agilent Technologies). Point mutations Figure 1. HIV-1 capsid assembly. The capsid protein (top right)
comprises N- (green) and C-terminal (red) domains.10During capsid assembly, the N-terminal domains form pentameric7(middle right) and hexameric11(bottom right) rings (with the N-terminal domains shown in blue and green, respectively, and the C-terminal domains shown in red in both oligomers). A model of the fully assembled capsid (left) comprises adjacent hexamers connected to each other via C-terminal domain dimers, and exactly 12 pentamers are required to close the cone.7
Figure 2.Summary of capsid constructs used in the current work. The delineation of the N- and C-terminal domains and the location of point mutations in the various constructs are shown. The dimerization states of the constructs under the experimental conditions used for NMR and SAXS/WAXS measurements are also indicated.
Journal of the American Chemical Society Article
dx.doi.org/10.1021/ja406246z|J. Am. Chem. Soc.2013, 135, 16133−16147 16134
tecture do incorporate such machinery and thus might form the basis for learning mechanisms that could account for our data [ J. E. Hummel and K. J.
Holyoak,Psychol. Rev.104,427 (1997)]. Our goal is not to deny the importance of neural networks but rather to try to characterize what properties the right sort of neural network architecture must have.
25. Supported in part by an Amherst College Faculty Re-search Grant to P.M.V. We thank L. Bonatti, M. Brent, S.
Carey, J. Dalalakis, P. Gordon, B. Partee, V. Valian, and Z.
Zvolenszky for helpful discussion, and P. Marcus and F.
Scherer for their assistance in construction of the test apparatus. We also thank Bell Labs for making available to the public the speech synthesizer that we used to create our stimuli. Some subjects in experiment 1 were tested at Amherst College; all other subjects were test-ed at New York University. The parents of all partici-pants gave informed consent.
11 September 1998; accepted 16 November 1998
Assembly and Analysis of Conical Models for the HIV-1
Core
Barbie K. Ganser,* Su Li,* Victor Y. Klishko, John T. Finch, Wesley I. Sundquist†
The genome of the human immunodeficiency virus (HIV) is packaged within an unusual conical core particle located at the center of the infectious virion. The core is composed of a complex of the NC (nucleocapsid) protein and genomic RNA, surrounded by a shell of the CA (capsid) protein. A method was developed for assembling cones in vitro using pure recombinant HIV-1 CA-NC fusion proteins and RNA templates. These synthetic cores are capped at both ends and appear similar in size and morphology to authentic viral cores. It is proposed that both viral and synthetic cores are organized on conical hexagonal lattices, which by Euler’s theorem requires quantization of their cone angles. Electron microscopic analyses revealed that the cone angles of synthetic cores were indeed quantized into the five allowed angles. The viral core and most synthetic cones exhibited cone angles of approximately 19 degrees (the narrowest of the allowed angles). These observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon. essen-tial roles in viral assembly: the NH2-terminal MA (matrix) region binds the membrane, the CA and NC condense about the viral RNA to form an unusual conical structure at the cen-ter of the virus (the “core”). The incen-terior of
the core is composed of an RNA/NC cylin-ders in the presence of RNA (5). Building on this, we screened for conditions that would mod-els for the viral core have suggested that the genomic RNA dimer dictates the cone mor-phology (8). The protein construct included both the CA and NC domains of HIV-1 Gag, because viral core morphology can be Lake City, UT 84132, USA. J. T. Finch, Structural Studies Division, UK. Medical Research Council
preparations of virions and synthetic cores were identical, and the two objects are shown at the same magnification.
RE P O R T S
1 JANUARY 1999 VOL 283 SCIENCE www.sciencemag.org 80
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The gag-‐pol polyprotein is cleaved into the PR, RT and IN. The PR is essential to mediate the processing of the gag and gag-‐pol precursors. The catalytic site of PR contains an aspartic acid (Asp) that is shared by a whole family of proteases. The enzyme active form starts to appear after the cleavage at the N terminus of the PR within the gag-‐pol and reaches a maximum level after the cleavage at the C terminus, a process that happens under acidic conditions. Indeed, artificially extending the N terminal domain of PR renders the virions unable to infect 65.
Upon maturation, the RT is found in the form of a heterodimer, composed of the two subunits p66 and p51 of 560 and 440 amino acids, respectively. The former subunit carries the catalytic domains of the polymerase and the RNase H activities 28.
The polymerase catalyzes the initiation of reverse-‐transcription and the elongation of the resulting transcripts, whereas the endonuclease mediates the gradual and subsequent degradation of the viral RNA templates. Recently, the crystal structures of the RT complexed with RNA-‐DNA and DNA-‐DNA duplexes has been solved and allows the understanding of how RT-‐inhibitors work 66,67.
The mature form of HIV-‐1 IN is a dimer that is composed of two catalytic core domains (CCD), via an interaction between the N-‐terminal domain (NTD) and the C-‐terminal domain (CTD). The active site of the CCD carries Asp and glutamic acid (Glu) that are both electronegative residues. Upon interaction with viral cDNA, two IN dimers come in contact, forming a tetramer 68,69. The Pre-‐
integration complex (PIC) that contains IN, binds to nuclear pore proteins as the Nup153, Nup160, RANBP2 and TNPO3, which allows it to be imported into the nucleus 70-‐74. Once there, the PIC targets the host genome via the interaction of IN with LEDGF/p75 that possess a chromatin-‐binding domain 75.
The lipid host-‐derived membrane contains bound env glycoprotein gp41 via non-‐covalent interactions. The TM protein gp41 interacts further with the other env-‐derived glycoprotein, namely gp120, which is in this way connected to the virion membrane and yet exposed to the surface (SU protein), as reviewed by
Wilen and colleagues 76. Heterodimers of gp41 and gp120 form further trimers that are the working module for viral fusion 77. Gp120 interacts with the specific CD4 receptor strongly expressed on the CD4+ T cells via mainly one of its five variable loops (VL1-‐5), VL3. This binding operates a conformational change that allows gp120 to bind one of two co-‐receptors depending on the cell type. CCR5 co-‐receptor is expressed on memory CD4+ T cells, Dendritic Cells (DCs) and macrophages. CXCR4 co-‐receptor is expressed on naïve and memory CD4+ T cells. The aforementioned conformational change allows additionally the gp41 monomer to expose the hydrophobic domain called the fusion peptide, representing the first step of the fusion between viral and host membranes 76.
The HIV-‐1 genome additionally codes for regulatory and accessory proteins. As reviewed by Karn and Stoltzfus 78, the two regulatory proteins are tat and rev that are required for proper HIV-‐1 provirus transcription and export of the resulting mRNAs from the nucleus to the cytoplasm, respectively.
Additional accessory proteins of HIV-‐1 are dispensable for the viral replication but assist in the evasion from innate and adaptive immunity. These are vif, vpr, vpu and nef. Vif and vpr both interact with cellular targets and cullin complexes to direct host defense factors to the proteasome 79. Vpu and Nef regulate the abundance of the cell surface molecules as CD4. Nef additionally targets other molecules expressed at the plasma membrane, as the CD3 receptor and Major Histocompatibility Complex (MHC) Class I molecules 79.
The structure of the HIV-‐1 genome is similar to that of other retroviruses in that it consists of two flanking LTRs with the previously mentioned conserved elements and a central coding region (figure 5). HIV-‐1 expresses one primary transcript that is either translated into a gag precursor (Pr55gag), a gag-‐pol precursor, or single/multiply spliced 78. Depending on the splice site used, the single splicing will result in the production of vif, vpr or vpu-‐env transcripts. Two different double-‐splicing events will result in the generation of the tat transcript and that of the bicistronic rev and nef 78.
Figure 5: Structures of the HIV-‐1 genome and virion. A) The HIV-‐1 genome is composed of two flanking LTRs containing typcial retroviral regulatory sequences, and different ORFs coding for gag-‐pol, env, the regulatory proteins rev and tat, and the accessory proteins vif, vpr, vpu and nef. The rev-‐responsive element (RRE, light blue box) is depicted below the env ORF (green box). B) The structure of the HIV-‐1 mature virion is depicted with the cleaved viral products, which are indicated with arrows. A) and B) from Sakuma et al, 2012 80.
1.1.8 The HIV-‐1 life cycle
The first step of the HIV-‐1 replication cycle is the viral particle fusion with the plasma membrane of a susceptible cell that expresses specific receptors. Upon the binding of gp120 to the CD4 receptor of an immune cell such as a
lymphocyte, it further interacts with a specific seven transmembrane domain G protein-‐coupled coreceptor depending on the virus tropism. Generally, CCR5-‐
tropic virus dominate the first phase of the infection, and gradually the tropism of some strains can change to reach up to 50% of viral particles that use the alternative CXCR4 co-‐receptor 81,82. Additionally, some strains of HIV-‐1 are able to use other coreceptors as CCR2b, CCR3, CCR8 and the orphan receptors V28, STRL33 and GPR15 83.
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