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HIV capsid assembly, mechanism and structure Bo Chen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00159 • Publication Date (Web): 13 Apr 2016 Downloaded from http://pubs.acs.org on April 14, 2016

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HIV Capsid Assembly, Mechanism and Structure Bo Chen Department of Physics, University of Central Florida, Orlando, FL 32816. HIV, Capsid Protein and Assembly. ABSTRACT: The HIV genome materials are encaged by a proteinaceous shell called the capsid, constructed from ~ 1000 - 1500 copies of the capsid proteins. Because its stability and integrity are critical to the normal life cycle and infectivity of the virus, the HIV capsid is a promising antiviral drug target. In this article, we review the studies shaping our understanding of the structure and dynamics of the capsid proteins and various forms of their assemblies, as well as the assembly mechanism.

Introduction Except the miraculous Berlin patient case, currently there is no cure for the Acquired Immune Deficiency Syndrome (AIDS) since it was first identified clinically in 1981. As its major pathogen, the Human Immunodeficiency Virus Type 1 (HIV-1) can trivially render all standard treatments and drugs fruitless with its fast replication and mutation rate. Relentless efforts have been directed to find its weak joints as more effective antiviral drug targets. The HIV capsid protein and capsid emerge as hopeful candidates1-5. Similar to all other viruses, the HIV genome materials are enclosed and protected by a proteinaceous shell called the capsid. In contrast to the symmetric icosahedral capsid, the HIV capsid is normally an asymmetric cone formed by ~ 1000 - 1500 copies of the capsid proteins6-8. In this article, to differentiate the capsid shell from the constituent protein, we will adopt CA as the abbreviation for the capsid protein, while referring to the capsid shell as the capsid. Preceding to the formation of the capsid, all CAs are part of the precursory structural proteins called gag, arranged in a spherical lattice filled with gaps and holes called the immature capsid7, 9. All the necessary viral components are present in the immature capsid, but the virus is non-infectious. The immature virus then has to go through a sequence of structural rearrangements termed maturation to become infectious10. During this maturation process, the cleavage of gag proteins by the viral protease produces isolated CAs. But only about 30% of the liberated CAs assemble around the ribonucleoprotein complex (RNP) to form the conical capsid7. Furthermore, the HIV capsid is much more than a convoy vehicle to protect and transfer the viral genome between infection sites. Different host factors interact with the HIV capsid to either assist or restrict the viral replication1, 3-5, 11-15. Mutations that lead to capsids of abnormal morphologies or altered stability usually attenuate or abolish the viral infectivity16-19. Although the mechanistic details are still not completely clear, evidence supports that the HIV capsid engages in both the reverse transcription of the viral RNA and the subsequent nuclear importation20-25. Therefore, manipulation of the structure and dynamics of the CA and capsid can be exploited to suppress the HIV. A number of artificial compounds were demonstrated to inhibit HIV infection by binding with the HIV capsid or CAs 1, 2, 5, 12, 15, 26. Naturally, the structure and mechanism of the HIV CAs and capsid assembly are of great interest and have been active fields of research in the past decades. Excellent reviews are available on different aspects of the HIV capsid1-5, 11, 14, 27, 28. In this article, we review the work that lead to our current understanding of the structure and assembly mechanism of the HIV capsid. Our discussion will start with the studies that elucidated the ACS Paragon Plus Environment

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structure and dynamics of the constituent HIV CA in the unassembled state. In the second section, we will revisit the work that defined the molecular arrangements and interactions in the HIV capsid and various in vitro CA assemblies. The third section will focus on the experimental work to probe the HIV capsid assembly process. The last section will summarize the theoretical studies and simulations of the HIV capsid assembly. In each section, our discussion will roughly follow the time-line of the research advancements, with brief comments and comparisons of relevant results. The structure and dynamics of the soluble HIV CAs

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Figure 1. Full-length structure and dynamics of the wild-type HIV CA. A. The HIV CA monomer as defined in 3J3Q.pdb . The NTD and CTD are colored in red and orange, respectively. All secondary structure components are labeled. B. The NTDs sample 64 a large conformation space around the rigid CTD dimer (white ribbons) in solution . The blue and red transparent regions demonstrate a reweighted probability at 50% and 10% of the maximum. C. 2D contour projection of distribution of the position of the NTD centroid relative to CTD. D. The structures of the protein corresponding to the six labeled clusters are shown in ribbon diagram, with the NTD labeled in green and CTD in white. Figures B to D are reprinted with permission from ref. 64. Copyright (2013) American Chemical Society.

The HIV CA shares a universal tertiary structure with the other retroviral CAs, despite little sequence similarity10. It is a 231-residue protein comprising two domains that can fold inde2 ACS Paragon Plus Environment

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pendently, linked by a short flexible inter-domain linker (P147, T148, and S149). Specifically, as shown in Fig. 1A, the arrow-head shaped N-terminal domain (NTD, residues 1-146) is composed of a short β-hairpin followed by seven α-helices; The ovoid-shaped C-terminal domain (CTD, residues 150-231) starts with a short 310 helix followed by four α-helices. Due to the high inter-domain mobility, it was challenging to crystalize the native HIV CA alone until very recently29. To circumvent this challenge, the two domains of the HIV CA were studied as separate entities initially. A crystal structure of the HIV CA NTD (residue 1-151) bound to the cyclophilin A (CypA) was first obtained by Gamble et al.30 CypA is a 165-residue cellular peptidyl prolyl isomerase that actively engages in the HIV infection activity via binding with the HIV CA31-40. The structures of two CypA and CA NTD in each asymmetric unit of the crystal are nearly identical. The helices 1-4 (H1-4) and H7 in the CA NTD are arranged into a bouquet of flowers: the helices are nearly parallel to each other with the bottom end (close to the flexible linker) bundled closer together. The short H5 and H6 are capped at the loose top of the helices bundle, in a nearly orthogonal direction to each other and to the rest of helices in the bundle. The N-terminus of the protein folds into a short β-hairpin, stabilized by a buried salt bridge between Pro1 and Asp51. This β-hairpin is not present in the precursory gag protein. Moreover, Pro1 and Asp51 are highly conserved in retroviral CAs41. Therefore they proposed that the formation of this βhairpin upon the proteolytic cleavage of the gag proteins initiates new interactions between CAs for the capsid assembly. This hypothesis is consistent with studies which showed that modification of the CA N-terminus can dramatically shift the in vitro capsid assembly morphology from the spherical immature capsid to tubular or conical mature capsid like particles42-45. The crystal structure shows that the CypA binding interface is stabilized predominately by van der Waals interactions and hydrogen bonds between residues 85-93 of the CA NTD, which is the long flexible loop between H4 and H531, 46, 47. Recent solid state NMR (ssNMR) and allatom molecular dynamics (MD) simulation results demonstrate that this CypA binding loop remains highly flexible in the tubular capsid assembly, but its flexibility significantly attenuates upon binding with CypA31. Furthermore, except for the residues in the CypA binding loop that exhibit some NMR signal shifts( < 2 ppm), the spectra of the CA tubular assembly display little change upon binding with CypA. Thus, it indicates that the overall CA structure remains unperturbed when binding CypA31. This is consistent with the fact that the structure of the CA NTD in the crystal30 appears nearly identical as that of the CA NTDs in the full-length HIV CAs determined recently by solution NMR46. However, Barklis et al. showed that the removal of residues 87-97 (∆87-97 CA) in the CypA binding loop promotes the in vitro CA assembly48: While the wild type (WT) capsid assembly normally demands high ionic strength (≥1 M NaCl), the mutant CA ∆87-97 can assemble in buffers with only 0.5 M NaCl into longer tubes. Combined, it indicates that the CypA modulates the CA assembly most likely through the perturbation of the CA dynamics rather than structure. Subsequent to the elucidation of the CA NTD structure, the dimer of the HIV CA CTDs was crystallized and its structure was solved by X-ray diffraction49. Two regions in the CTD are note-worthy. The first is the dimer interface, centered around Trp184 and Met185 at the second helix of the domain (or H9 in the complete CA). Mutation of Trp184 or Met185 causes the CA to abolish dimerization. The dimerization strength of the truncated individual CTDs was measured to be Kd ~ 10 µM49, while the full-length CAs exhibit a Kd ~ 18 µM49-51. The second important segment is the 20-residue segment towards the N-terminus of the CA CTD, the so called major homology region (MHR, residues 153 to 172)52, which is highly conserved in all retroviral CAs53, 54. This region folds into a 310 helix followed by an α-helix49. In addition, a number of hydrogen bonds between the conserved residues in MHR stabilize the overall packing of the four helices in the CA CTD. Mutations of residues in MHR impair the HIV CA assembly and infection53, 55-58. Similar effects were found in other retroviruses, yet sometimes the ACS Paragon Plus Environment

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detrimental mutations in MHR can be rescued by a second mutation away from MHR59-61. Although this MHR is not part of the dimer interface at the CTD, nor does it belong to any interface in the mature CA assembly, mutagenesis studies show modifications in MHR perturb strongly the dimer affinity59, and the MHR contributes to the intrahexamer interface in the immature capsid lattice9. The last 11 residues at the CA CTD terminus appear to be completely disordered, confirmed later to be highly dynamic in both soluble and assembled state31, 62-64. Subsequently, various structural models of HIV CA NTDs and CTDs in different forms were obtained by different techniques. In general, the CA NTD appears to adopt a similar fold 30, 46, 65-69 . However, the CA CTD can form dimers in slightly different conformations49, 65, 68, 70-74. Except for the domain swapped form of the CA CTD dimer where MHR becomes the major dimer interface71, the general fold of the CA CTD is nearly the same in these dimers49, 64, 65, 68, 71, 72, 74 as that in its monomeric state66, 70, 73, 75-77. However, the crossing angle between H9 at the dimer interface differs. This plasticity of the dimer interface may contribute to the formation polymorphic HIV CA assembly. However, in these studies, either mutations were introduced to residues at the dimer interface71, 76, or the structural models were solved in the presence of other molecules65, 67-69, 72, 77-79, either of which may perturb the dimer interface artificially and cause the different dimer interface. Combining solution NMR with small and wide angle X-ray scattering(SAXS/WAXS), Deshmukh et al. overcame the challenge of NMR line-broadening of residues at the linker and dimer interface and determined the structure and dynamics of the full-length HIV CA in the soluble state64. Despite the molecular motion, the structures of individual domains remain unchanged and they were treated as rigid bodies tethered by the flexible linker in the structural characterization. By measuring the HIV CA in different alignment mediums, the shape and orientation of domains were unambiguously determined by the residual dipolar coupling (RDC) constants between the amide nitrogen and proton atoms, and the size and shape of the protein were defined by the SAXS/WAXS data. In addition, the monomer-dimer equilibrium in solution was accounted for in the ensemble simulated annealing simulations. They showed that the full-length HIV CAs maintain a rigid CTD dimer in the same conformation as the truncated CTD dimer construct (residues 144-231)80. Therefore, different dimer interfaces observed in ref. 49 and 74 are likely due to crystal packing. However, their method does not rule out completely the possibility of a small dimer population present in other conformations. On the other hand, the two CA NTDs undergo constant large-amplitude flip-flop motions relative to the rigid CTD dimer enabled by the flexible inter-domain linker, but exhibit no stable inter-domain interactions. They further quantified the conformation space sampled by the inter-domain flip-flop motions of the NTDs, shown in Figs. 1B to D. By fitting the HIV capsid cone model81, they showed that the rigid CTD dimer plus flexible NTD to CTD orientations sampled in solution are sufficient to accommodate the curvature variations on the capsid surface between pentameric and hexameric CA assemblies. However, only less than 2% of the populations in their ensemble exhibit the conformation consistent with those identified in hexameric CA assembly8, 80, 82-84, while 5% of the population adopt the conformation resembling that identified in the pentameric CA assembly 81. Obviously, this does not agree with the fullerene motif where predominant HIV CAs form hexameric assemblies in the HIV capsid6, shown in Figs. 2A and B, explained in the next section. This discrepancy was ascribed to the concentration dependent effects of the protein dynamics, as the assembly is normally initiated by oligomerization of the highly concentrated HIV CAs via crowding effect85 and19 high salt86. The structure and dynamics of the HIV CAs in assemblies

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Figure 2. Structural models of various HIV CA assemblies. A. and B. The fullerene motif to create various shaped assemblies from p6 hexameric lattice with pentamer insertion. To create a cone from a p6 hexameric lattice, a continuous patch of hexamers can be removed between the vertical and the other arrow-headed lines (P =1 to 5), and seal the remaining lattice together without

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disruption of the lattice, as shown at the top of A. By doing so, the tip angles of the cones created will only have five possible values: 112.9° of P=1, 83.6° of P=2, 60° of P=3, 38.9° of P=4, and 19.2° of P=5, as shown at the bottom of A. Then, according to Euler’s theorem, exactly 12 pentamers are also needed to close the cone, and their positions in the hexameric lattice will determine the assembly shape. A fullerene cone with 19.2° tip angle is shown on the left of B, with seven pentamers distributed at the broad end and five pentamers at the narrow tip of the cone, resembling the typical authentic HIV capsid. Alternatively, a symmetric spherical assembly can be obtained if the pentamers are uniformly dispersed in the hexameric lattice, shown on the right of B. All pentamers in B are shaded. C. and D. The structural models of the WT HIV CA hexamer in hydrated and dehydrated states, 29 according to pdb 4XFX and 4XFY , respectively. E. The structural model 3P05.pdb of an isolated pentamer formed by mutant 81 HIV CAs with disrupted dimer interface . Molecules are plotted in ribbon model, with each subunit in a different color. The dots in C and D are water molecules in the crystal. F. All-atom model of the HIV capsid generated combining cryoEM and MD simulation. All CTDs are colored in red. All NTDs in hexamers and pentamers are colored in blue and green, respectively. G. Interactions between a dimeric HIV CAs and the neighboring subunits in a flat 2D hexameric lattice. The hexameric assembly is 83 based on 3H47.pdb . The dimer interface between H9 is constructed based on 2KOD.pdb. The CTDs and NTDs of the dimer are plotted in orange and red, respectively. The CTDs and NTDs of their neighbors are plotted in green and cyan for distinction. The NTD-NTD, NTD-CTD, and the trimeric interfaces are shown in zoom-in views in indicated regions from H to J. The closest helices at each interface are identified, with solid lines connecting the points of closest distances between their symmetric axes. Figures A and B are from ref. 6. Reprinted with permission from AAAS. Copyright (1999) AAAS. Figure F is adapted with permission from ref. 8. Copyright (2013) Nature Publishing Group. Figures G to J are adapted from ref. 143. Copyright (2015) Elsevier Ltd.

Many viral CAs can self assemble in vitro into structures similar to their authentic in vivo capsids. In 1992, Ehrilich et al. demonstrated for the first time that the recombinant HIV CAs can assemble into tubes in 1 M NaCl pH 6 buffer. Although this tubular assembly differs from the in vivo conical capsids87, it implies that the CAs may play an essential role to stabilize both the immature spherical and the mature conical capsids. In addition to electron microscopy (EM) analysis of the assembly morphology, they identified the most stable oligomers to be the CA dimers by analytical ultracentrifugation and nondenaturing gels. Then Gross et al. and Campbell et al. showed that the presence of the nucleocapsid protein (NC) tagged to the CA Cterminus can further reduce the required protein concentration for assembly by as much as 20fold with the addition of RNA88, 89, indicating the electrostatic interactions between NC and RNA can promote the nucleation of CA assembly. Ganser et al. further confirmed that the CANC assembly does not depend on the RNA sequence6. In addition, they proposed that the conical HIV CA assembly is formed by insertion of 12 CA pentamers into the CA hexameric lattice, based on the quantization of the conical tip angles of the HIV capsids and the resemblance to the carbon Buckminster fullerene90, as shown in Figs. 2A and B. The first evidence confirming this fullerene hypothesis was soon presented by Li et al.91 with the 20 Å resolution cryo-electron microscopy (cryoEM) model of the in vitro tubular assembly. By docking the high resolution structures of individual domains on to their cryoEM electron density map, they demonstrated for the first time that the tube is constructed by the CA hexameric lattice91. Briggs. et al. then applied cryoEM to image the mature capsids directly isolated from the HIV particles, and demonstrated that authentic HIV capsids exhibit a similar hexameric symmetry in the in vitro tubular assembly. In a similar approach, Benjamin et al. analyzed the authentic HIV capsids and showed that the majority of isolated cores display a conical tip angle around 19°92, consistent with the prediction of the fullerene hypothesis 6. However, the specifics of intersubunit contacts could not be resolved in these studies due to the limited resolution. Meanwhile, a number of mutagenesis studies18, 19, 93, hydrogen-deuterium exchange94 and mass spectroscopy analysis95 suggested that the assembly interfaces exist between the CA NTDNTD at H1 and H2, and between NTD-CTD at H4 and H10, in addition to the dimer interface between H9 at the CTD. The direct resolution of specific interfaces was obtained by Ganser et al. in their pseudoatomic structure model of the flat hexameric lattice of the HIV CAs by cryoEM84. They were able to obtain a 9 Å resolution density map of the hexameric assembly of flattened large spheres (radius ~ µm) assembled with the R18L mutant HIV CAs. By docking high resolution structures of individual domains, they resolved three specific intersubunit assembly interfaces in the HIV CA hexameric assembly, down to the helix level. The NTD-NTD interface between H1-3 of each subunit binds six adjacent subunits at the very center of each hexamer, shown in 6 ACS Paragon Plus Environment

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Figs. 2G and J. The hexamer is further stabilized by the NTD-CTD interface between H4 and the groove between H8 and H9, as well as the loop between H10 and H11, shown in Figs. 2G and H. The dimer interface between H9 connects hexamers into a lattice. All the resolved interfaces confirmed earlier implications from biochemical and biophysical analyses18, 19, 93-95, and are consistent with the structural studies from other similar retroviral CA assemblies61, 96, 97 . However, the six β-hairpins of the subunits in the same hexamer do not form any close contacts, different from those in the hexamers formed by the CA NTDs of the N-tropic murin leukemia virus (N-MLV)97. It indicates potential structural differences at the quaternary level in different retroviral CA assemblies despite the similar tertiary structures of subunit CAs. Subsequently, additional results from M. Yeager’s group quickly advanced the structural models of the HIV CA hexameric and pentameric assemblies to atomic resolution81-83. They were able to crystalize isolated hexameric and pentameric assemblies with mutant HIV CAs incorporating cysteine crosslinking82, 83 or fusing the CAs with CcmK4 proteins83. At this improved resolution, a large number of water-mediated hydrogen bonds between the polar side chains and backbone atoms were shown to be around the hydrophobic center of the NTD-NTD interface. These water-mediated hydrogen bonds are able to reposition themselves to accommodate the variations between slightly different pentameric and hexameric assemblies with a conserved hydrophobic core at the NTD-NTD interface. In addition, the charged residues are positioned at a closer distance at the center of the pentameric assembly, implying that electrostatic interactions may be the switch to form the quasi-equivalent assembly, shown in Fig. 2E. By comparing the structures of different hexameric crystals, they showed that the structural differences can be recapitulated by varying relative orientations between NTDs and CTDs without changing the structure of individual domains83. An atomic model of the conical HIV capsid was constructed using the high resolution models of isolated hexamers and pentamers as building blocks following the fullerene motif81. In this process, it appears that different dimer interfaces are needed to induce the variable curvature on the capsid surface81. However, their hexamer and pentamer structural models do not contain any dimer interface due to the disruptive mutations81-83. In addition, Byeon et al. solved a 16 Å resolution model for the tubular assembly of the HIV CAs by combining cryoEM and the CA CTD dimer model of solution NMR80. The CTD dimer structure determined by solution NMR fits well with the dimers in their tubular assembly cryoEM density map, and agrees reasonably well with the 2D crystal of flattened spherical assembly84 except for the different crossing angle between H9. This solution NMR dimer interface is essentially identical to the rigid dimeric interface in the full-length CAs in soluble state determined by solution NMR and SAXS/WAXS64. In addition to the three interfaces identified by Ganser et al. in their hexameric model83, 84, a new trimer interface was identified. This trimer interface is formed by H10 and H11 at the three-fold axis of adjacent hexamers to stabilize interhexameric interactions80, shown in Figs. 2G and I. The absence of this interface in previous 2D crystal structure84 and its presence in the tubular assembly suggests its role in curvature formation. In their subsequent work, the resolution of the tubular assembly model was improved to 8 Å8. Details of the trimeric interface was resolved: a hydrophobic interface at H10 including residues Ile201, Leu202, Ala204, and Leu205 was shown to be the core of this interface. Additional mutagenesis and infectivity tests, and coarse grain simulations confirmed this observation. Furthermore, at this improved resolution, they found the CA NTDs in the tubular assembly share a common fold, but the CTD dimer interfaces exhibit somewhat flexible crossing angles between H9 (36°, 44°, or 54°). In addition, based on this tubular model, atomistic models of various conical HIV capsids were generated by combining all-atom MD simulations81, as shown in Fig. 2F. All-atom MD simulations of the complete cone showed that the trimer interface exhibits the largest differences to reconcile pentamer and hexamer con-

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tacts, implying that the trimer interface is the critical determinant for the curvature variations on the conical HIV capsid surface. At this point, there is no consensus regarding the cause of the variable curvature of the conical HIV capsid. The general agreement is the folds of individual domains are largely conserved upon the assembly. This was confirmed by ssNMR studies of the tubular HIV CA assemblies63, 98 . SsNMR studies also revealed that residues at the CA N, C termini and the CypA binding loop between H4 and H5 remain highly flexible in the assembly31, 62, 63. In addition, the flexible linker exhibits millisecond scale motions that could induce variations of inter-domain orientations to contribute to the assembly polymorphism 99. In 2015, Gres. et al. finally overcame the challenges to crystallize the hexameric assembly of native HIV CAs and solved the atomistic structure by X-ray diffraction29. Their results largely agree with the four interfaces identified in the isolated hexamer models formed by mutant HIV CAs82, 83, the 2D crystal84 and tubular assembly8, 80. However, the dimer and trimer interhexamer interfaces are much closer in their crystal structure than those shown in the hexamers formed by mutant HIV CAs83, or those binding with antiviral peptide/ligands13, 100. The most striking outcome is the comparison of the hexameric assembly at hydrated and de-hydrated states, shown in Figs. 2C and D: controlled hydration variation can cause 3 to 6% variations of the unit cell dimension of the crystal, which arises mostly from the tighter trimer and dimer interfaces by a closer packing at the dehydrated state. The structural changes are enabled by the adjustment of the pervasive water mediated hydrogen bonds at these interfaces. However, it remains to be proved if similar mechanisms are employed by the HIV CAs to induce the variable curvature in the in vivo and in vitro assemblies. Recently, Valbuena and Mateu assessed the mechanical properties of the 2D sheet assembly of the HIV CAs by Atomic Force Microscopy (AFM)101. The HIV CAs readily form 2D lattice in near physiological conditions(phosphate-buffered saline), both in solution and on the mica surface. Operated in the liquid mode, the AFM provides sufficient imaging resolution to resolve the height differences of different domains in the assembly. The height histogram shows that the hexameric assemblies of the negatively charged NTDs lay on top of the CTDs in the lattice, while the positively charged CTDs attach to the negatively charged mica surface. The average distance between neighboring hexamers was measured to be 9.9 nm, closely matching the 9.3 nm value measured by cryoEM84. By measuring the variations of distances between neighboring hexamers, they showed that the lattice exhibits a breathing motion with 1.34 nm amplitude at 25 °C. Transient defects were observed in the lattice, arising from the disruption and re-association of the weak NTD-NTD/NTD-CTD contacts. Moreover, the spring constant (0.44 N/m) of the lattice calibrated by the indentation experiments agrees well with the value (0.39 N/m) derived from coarse grain (CG) simulations102. When the indentation was extended out of the elastic regime, the rapture of the lattice displays a two-step process, associated with the disruption of the top weaker NTD assembly first, and followed by the higher force step to break down the stronger lower CTD-CTD interface. Remarkably, the ruptured hole can self-heal completely, if it is away from the lattice boundary, via a two-step process reversing the disruption of the lattice. Furthermore, lattices assembled in the presence of betaine, a small chemical capable of binding HIV CAs, appear indistinguishable from those assembled without betaine. However, betaine binding greatly enhance the dynamic motion of the lattice as well as the elasticity. This is likely due to the binding of betaine to the hydrophobic residues at the NTD-NTD, NTD-CTD, and trimer interface. If this were true, the HIV CA assemblies formed in the presence of CypA should be more brittle, as the binding of CypA reduces the mobility of the loop between H4 and 531, thus restricts the flexibility of the NTD-NTD and NTD-CTD interface. In addition to the structural studies of the HIV CA assemblies, considerable knowledge was learned from assemblies formed by the CAs of other retroviruses. Cardone et al. and Hyun et ACS Paragon Plus Environment

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al. established structural models of the capsids of the Rous Sarcoma virus (RSV) in T = 1 and 3 icosahedral assemblies103, 104. Their results lent support to the existence of pentameric CA assemblies in the fullerene motif6. The plasticity of RSV CA assemblies was further demonstrated by a series of work59, 60, 104-112. These assemblies were stabilized by similar interfaces as those in the HIV CA assemblies103, 106, supported by a number of biochemical and mutagenesis experiments59-61, 108, 109, 111. However, they also display notable differences: while the HIV CAs exist as dimers in solution and assemble at neutral pH and high concentrations of NaCl, the RSV CAs are monomeric in solution and could not assemble in the NaCl buffer. Instead, the nucleation of the RSV CA assembly is initiated by acidic pH104, 105 or addition of phosphate buffer111. The RSV CAs form a different dimer interface104, 105 and undergo different assembly kinetics111. More significant differences were observed at the quaternary level in the hexamer of N-MLV97 and the bovine leukemia virus (BLV) CAs113, whose CAs share a common tertiary fold as the HIV CA despite little sequence overlap. Different intermolecular contacts and inter-domain arrangements of the CAs were identified in the immature capsids of different retroviruses9, 114. Collectively, it seems that a wealth was learned regarding the nonicosahedral and polymorphic CA assemblies, but to completely decipher how and why retroviral CAs of conserved 3D fold form distinct capsids, more structural studies of different retroviral CA assemblies at atomistic resolution are needed. The experimental studies of the HIV CA assembly mechanism

Figure 3. CryoEM images of authentic HIV capsids. A. and B. Tomographic slice through a viral particle exhibiting curling sheet and a capsid with an extra sheet to its left side, respectively. C. Simulated structure of curling sheet in a roll. D. TEM images showing normal virions and abnormally large virions with free floating multi-capsids. The black scale bar is 500 nm. E. 3D rendering of tomograms of a typical multi-capsid virions. Figures A-C are adapted with permission from ref. 120. Copyright (2013) Elsevier Ltd. Figures D and E are adapted with permission from ref. 124. Copyright (2015) Nature Publishing Group.

The in vivo assembly of the HIV capsid takes place as a part of cascading events. Due to the complexity, it is difficult to directly monitor the capsid formation during this process in real time. Alternatively, many experimental studies probed this process with snapshots of EM and cryoEM. Early analysis by Gelderblom showed that the viral RNP are consistently packed towards the broad end of the capsid, while the narrow tip appears empty115. Hoglund et al. fixed the HIV infected cells and obtained clear images of HIV particles by negative-staining116. The mature capsids seem to always span the entire viral membrane envelope, with the narrow tip attached to the viral envelope. Subsequently, Welker et al. designed a method to strip away the viral envelope to directly image the authentic HIV capsids by EM117. They showed that the angle of the narrow tips averages around 21.3°, in spite of the heterogeneous viral capsids. In 2003 Briggs et al. performed cryoEM analysis of the authentic HIV capsid with the viral envelope stripped away118. Majority of the capsids were conical shaped with considerable polymorphism. The narrow tips exhibit an angle of 19.7°, consistent with the fullerene motif6. A minority (7%) of the capsids were tubular shape, allowing easier imaging analysis. Under diffraction, these tubular capsids display a regular spacing of ~ 9.6 nm with six-fold symmetry, consistent with the previous EM analysis of the in vitro HIV CA tubular assembly91. Therefore, this ACS Paragon Plus Environment

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work justified the clinical relevancy of structural studies of the in vitro HIV CA assemblies in the place of the more complicated in vivo capsids. Benjamin et al. conducted cryoEM analysis of the authentic HIV capsids in intact viral envelopes92. Around two-thirds of their samples display the typical conical shape, with the tip angle between 18° to 24°, consistent with the fullerene motif6. The capsid size exhibits considerable heterogeneity. To better resolve the conserved features at the limited resolution, they aligned the tip and broad base end of the capsids respectively and found a consistent 11 nm gap between the viral envelope/matrix layer and the capsids. In addition, many capsids appear to have a hole at the narrow tip end. Based on these observations, they proposed that the CA assembly is a de novo process rather than a gradual evolution from the immature capsid without disassembly. They also observed extra density inside the broad end of the HIV capsids, attributed to the viral RNP. It is possible that the viral RNP interact with the CAs to nucleate the assembly as a template. However, the observation of multiple capsids inside a single virion indicates that the CA assembly could also be a concentration driven process. In 2006, Briggs et al. performed cryoEM on authentic unstained HIV particles. Roughly 63% of their capsids are conically shaped, a ratio similar to the statistics in ref. 92. The capsids always span the entire virion despite the large virion size variations (diameter from ~122 to ~158 nm), in agreement with ref. 116. The narrow tips of the capsids have an averaged diameter of 27 nm with an angle ~ 19.2°, independent of the virion size, while the broad ends span a diameter ~ 56 nm exhibiting a strong correlation with the virion size. They observed additional density inside the capsids towards the broad end, with an 8 nm spacing from the capsid layer. This separation is consistent with the gap between CA and NC in the immature lattice119. It also agrees with what Benjamin et al. observed earlier92. On the other hand, while the broad end appears completely closed and is consistently separated at ~12 nm away from the viral membrane, the narrow tip of the capsids is closer to the viral membrane, and sometimes displays visible density attached in between, in contrast to earlier observations92, 115, 116. This difference was ascribed to the improved imaging technique and virion preservation. Based on these observations, Briggs et al. proposed that the HIV CA assembly starts from the narrow tip end, and terminates by the interactions with RNP and constrained by the viral membrane at the broad end. In an alternative approach to probe the assembly mechanism, Yu et al. analyzed the failed CA assemblies in the authentic HIV and Equine Infectious Anemia virus (EIAV) by cryoEM120. Interestingly, they found curved sheets in rolls, shown in Fig. 3A. These curling sheets can coexist with complete capsids, shown in Fig. 3B. They appear to be incomplete capsids arrested in intermediate states towards the final assembly. At the resolution, individual hexameric assemblies could be resolved on the capsid surface. Remarkably, among ~ 20% of all capsids, they found holes aligned on the capsid surface, indicating possible misalignment of the rolled sheet to complete the capsid assembly. These holes are sufficiently large for the green fluorescent protein to transverse. However, it is not clear if these structural defects impair the viral infectivity. In summary, their observations suggest that the CA assembly is derived from curved hexameric sheets. Their results are consistent with the irreversible growth model of the nonequilibrium continuum theory of elasticity121, 122, shown in Fig. 3C. More details of the simulation model will be explained in the next section. All of the viral image analyses aforementioned support the view that the HIV CA assembly is a de novo process driven by concentration or guided by interactions with RNP as a template following the complete disassembly of the immature capsid lattice92, 120, 123. However, more recently, Frank et al. presented evidence challenging this view124. In their high resolution cryoEM analysis, they observed multiple HIV capsids of normal size (~ 120 nm in diameter) present in many single super large (200 to 800 nm in diameter) non-spherical viral membrane envelopes, shown in Figs .3D and E. The variable surface area ratio of the viral membrane envelope to ACS Paragon Plus Environment

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capsid excludes the possibility that these large particles are derived from fusion of multiple virions. On the flat portions of the viral envelope membrane, patches of matrix protein (MA) lattice could be resolved. These MA lattices assume the same arrangements identified in earlier studies of the trimeric MA lattice on lipids125. In addition, some capsids were found in the intermediate states of assembly, attached to the membrane envelope in a side-way position, shown in Fig. 3E. These membranes are thicker and display the structural characteristics of typical immature capsid lattice, hugging the thinner mature capsids. Individual pentameric and hexameric assemblies were resolved on the capsids, as well as other structural defects including heptamer and gaps. The structural defects were predicted in theoretical modeling120, 121 and coarse-grain simulations of HIV CAs assemblies126. Taken together, this work supports the view that the mature HIV capsids arise from a gradual non-diffusional transition of the immature capsid lattices. Meanwhile, the in vitro HIV CA assembly were studied to probe the in vivo capsid formation. It allows more controlled experimental observation. Lanman et al. was the first to track the in vitro HIV CA assembly kinetics by optical turbidity93. The HIV CAs typically assemble into tubes with length up to micrometers, which produce sufficient light scattering for a qualitative assessment of the assembly kinetics by turbidity. The turbidity data demonstrates that the HIV CA in vitro assembly kinetics exhibits the typical sigmoidal profile similar to that of icosahedral capsids127: the process starts with a nucleation-limited lag time followed by a rapid elongation. Results of Lampel et al. in a later study confirmed this sigmoidal assembly kinetics128. Moreover, they found that addition of separated CA NTDs or CTDs hampers the efficient assembly at pH 7, but addition of separated NTDs suppresses the inhibition effect of isolated CA CTDs at pH 8. However, these studies did not identify the assembly nucleus. Del Alamo et al. used the in vitro CA assembly with inert macromolecules to probe the crowding effect on the in vivo CA assembly due to the presence of other viral and host components in the restricted viral envelope85. They showed that addition of inert crowding agents lowered the required ionic strength to near physiological salt concentration (~ 150 mM), and reduced the minimum CA concentration by over two-fold with no apparent effect on the assembly morphology. In the presence of crowding agents, the assembly appears reversible, while the process in high salt condition exhibits little reversibility. Moreover, the crowding effect allows the assembly of mutant CAs with attenuated dimer interface. Their later studies showed that the crowding effect cannot rescue mutant proteins with completely disabled dimer interface129. In summary, the inert physical crowding effect tends to promote the assembly of HIV CAs. Potentially, interactions with other parts of the viral and host materials may also contribute to the HIV CA assembly, for example, the electrostatic interactions between the lipids and HIV CAs can suppress the assembly130. Barklis et al. used a novel fluorescence microscopy (FM) method along with EM to directly monitor the assembly growth at micrometer resolution and analyzed how different factors affect the WT and ∆87-97 CA mutant assembly48. They showed that the assembly is most effective in high salt buffer at neutral pH. A short incubation at 37° spurs more nucleation of assembly. In addition, deletion of residues 87-97 in the CypA binding loop promotes the assembly of longer tubes in buffers with 50% lower salt concentration than that for the WT CAs. Since the WT and ∆87-97 CAs can co-assemble, by exploiting the immunofluorescence of specific antibodies for WT CAs, they showed that the tubular assembly has no preference of direction. The theoretical studies of the HIV CA assembly mechanism Theoretical studies of the HIV CA assembly benefit greatly from a suite of techniques and methods established for the icosahedral viral capsids127, 131. Specifically, the continuum theory of elasticity explained the shape transition of icosahedral capsids by the competition between the in-plane stretch (variations of distances between ACS Paragon Plus Environment

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neighboring subunits) vs. the out-of-plane bending energy (abrupt change of curvature such as vertices)132. Nguyen et al. extended this theory to describe the non-icosahedral capsids of retroviruses133, 134. To do so, they introduced a contribution that depends on the preferred curvature of the capsid, termed the spontaneous curvature, to the system Hamiltonian. Combined, their theory predicted a transition of the assembly shape from tubular to conical by minimizing the Hamiltonian with a volume constraint. The constants in the Hamiltonian can be estimated experimentally. The stretching and bending moduli could be estimated by CG simulations, or the capsid deformation experiments by AFM101. The spontaneous curvature can be determined by analyzing the capsid shapes by cryoEM. Thus, their theory combines microscopic and macroscopic properties of viral capsids to explain the HIV capsid shape. The volume constraint appeared to be true based on the available cryoEM analyses of the authentic HIV capsids at that moment92, 116, 118, 123 : the capsids seem to always span the entire viral envelope. Recently, Frank et al. observed free-floating HIV capsids of normal size in the extraordinarily large viral envelope124, which suggests that this volume constraint to minimize the energy may not be applicable, or at least may not be the only constraint that should be applied. Based on a similar frame work to that of Nguyen et al.133, 134, Hick and Henley demonstrated the construction of irregular capsids in terms of equilateral triangular shaped subunits with a set of irreversible growth rate reminiscent of the local rules theory of icosahedral capsids121, 135-137 . At each growth step, the assembly intermediate samples a range of “conformations” around the energy minimum with variable local geometric vacancies to add subsequent subunits. The addition of a subunit is then selected by a rate that depends both on the local geometry of the available vacancies and the global energy minimum of the assembly intermediate. They showed that the capsid size is predominantly determined by the spontaneous curvature. A number of the failed modes observed in their simulations agree well with recent cryoEM images of structural defects (holes, heptamers) on authentic HIV capsids120, 124. Levandovsky and Zandi et al. took a slightly different approach than that of Hicks and Henley to construct the irregular capsid using subunits as triangular prisms122. The tapering shape of the prism defines the intrinsic spontaneous curvature. Due to the weak binding between subunits, they argued that the addition of subsequent subunits to an incomplete capsid is to maximize the contacts and to minimize the energy at the given spontaneous curvature. They showed that the conical HIV capsid can be obtained as an intermediate state between the spherical and tubular assembly in the phase diagram, selected by the spontaneous curvature. Based on this framework, they could accurately simulate a range of intermediates such as unclosed capsid sheets wrapped in rolls or curved sheets as identified by cryoEM120, shown in Fig. 3C. A second approach to understand the HIV CA assembly is by CG simulations. Krishna et al. showed that the assembly of a stable HIV capsid with the correct hexameric symmetry requires an accurate representation of the directional interactions between subunits, e.g., the CG model with at least eight-sites/dimer138. Specifically, each NTD has to be described by at least a triangular shape resembling the three helices bundle from each subunit at the center of each hexamer83, 84. While the NTD-NTD interactions coalesce subunits into flat lattices of hexameric symmetry, they showed that directional interactions between NTD-CTD and CTD-CTD are responsible to induce the curvature observed in tubular and conical HIV capsids. Subsequently, Bo Chen and Tycko designed a novel CG model to simulate the HIV CA assembly that captures the backbone structure of the HIV CA126. As the protein consists mostly of α-helices, this model accurately represents the protein backbone structure by a group of cylinders as a rigid body, shown in Fig. 4A. The intersubunit interactions are represented by angular restrained Lennard-Jones potentials similar to that used in ref.139 to phenomenologically mimic the attractive interactions between the close contacts identified in structural studies84. The higher computational cost due to the high resolution limited their simulations to systems of 36 dimeric subunits in 2D. Nonetheless, this model can correlate molecular structure details to ACS Paragon Plus Environment

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the assembly mechanism that could not be resolved at a lower resolution. Specifically, they predicted that the trimers of dimeric subunits are the most stable and populated oligomeric intermediates during the assembly process. We note that this prediction was based on simulations incorporating only the NTD-NTD and NTD-CTD interfaces identified in the 2D flat lattice of the HIV CAs84. It agrees with the trimer interface identified in the tubular assembly80. Later, trimers were directly identified by AFM in the HIV CA assembly formed on lipids140, and kinetics modeling of the turbidity assay of the HIV CA oligomerization also indicates that trimers are the critical assembly intermediates141. Collectively, they lend justification of the triangular subunits used in the irreversible growth model of the capsid120-122. In addition, pentamers and heptamers were transiently observed. However, isolated hexamers were shown to be unable to seed the assembly. Instead, the assembly is driven by elevated concentrations. The optimal concentration for assembly determined by the simulations agrees qualitatively with experimental results19, 86. An enhanced version of this model with 3D rotational motions and 2D translation were used later to probe contributions of interactions at specific interfaces to the assembly and its results agree qualitatively with the mutagenesis analysis8. Subsequent to this cylindrical CG model126, Grime and Voth designed a second high resolution CG model for the HIV CA142. This model uses beads to represent each Ca atoms in the αhelices, and potentially is able to probe the CA assembly at an even higher resolution. However, a weak harmonic potential was applied to restrain all dimeric subunits to a plane or spherical surface, which limits its ability to probe the interesting question of the retroviral CA assembly: polymorphism and curvature formation. Consistent with the earlier 2D simulations with the cylindrical model126, trimers of dimeric subunits were shown to be the most populated oligomeric intermediates. In contrast, they found stable pentamers in their simulations. In addition, they showed that a flexible dimer interface would enhance the population of trimers and hexamers, but suppress the pentameric assembly as the system stabilizes.

Figure 4. 3D CG simulations of HIV CA assemblies by the cylindrical model. A. The cylindrical model of the HIV CA based on 8 3J3Q.pdb , capturing accurately the protein backbone structure shown in Fig. 1A. B. A curved hexameric lattice with 65.2 nm diameter assembled by a system of 128 dimeric subunits with variable NTDs to CTDs orientations derived from all-atom MD simulations. C. A sharp curvature assembly with 36.5 nm diameter formed by quasi-equivalent hexamer and pentamers, high81 lighted in blue polygons. The simulation system consists of 128 dimeric subunits based on 3P05.pdb (pentamer structure) and 8 2KOD.pdb (44° crossing angle of H9 at dimer interface). D. A sharp curvature assembly with 40 nm diameter formed by tetramers and a pentamer, highlighted in blue polygons. The simulation system consists of 128 dimeric subunits based on

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3P05.pdb (pentamer structure) and the dimer interface modeled based on chains 7 and S in 3J34.pdb (34° crossing angle of H9 at dimer interface). E. to G. are the assembly pathways derived by the average of ten simulations with identical setup as in B to D, respectively. The dimeric subunits used in the simulations are shown in the inset of the figures. NTDs and CTDs are colored in red and orange, respectively. Figures adapted from ref. 143. Copyright (2015) Elsevier Ltd.

Recently, Xin et al. enhanced the cylindrical model to simulations of larger systems (up to 256 dimeric subunits) with complete 3D freedom. This advancement enables the simulations to probe the mechanism governing the polymorphism and curvature control of the HIV CAs at a molecular level143, 144. In addition to accurately capture the protein backbone structure, a static ensemble of subunits in a range of conformations were used to mimic the variations of interdomain orientations sampled by subunits due to protein dynamics64, shown in Figs. 4B and E. They showed that hexameric lattices with heterogeneous and mild curvatures (radius ≥ 50 nm) were produced consistently, as shown in Fig. 4B. It suggests that protein dynamics is the cause for the polymorphic assembly. The assembly of these hexameric lattices were born from a large population of trimers, as the assembly pathway of such systems shown in Fig. 4E, similar to previous simulations in 2D126 and semi 3D8, 72. Isolated hexamers were shown to be unstable to act as the assembly nucleus. To assemble highly curved structures, it demands subunits to adopt conformations based on the pentameric template81. The resulting assemblies exhibit curvature nearly the same to the narrow tip of the authentic HIV capsid123, with the typical quasi-equivalent coexistence of pentamers and hexamers, shown in Fig. 4C. They further showed that a small deviation (10°) of the crossing angles between H9 at the dimer interface from the experimental value64, 80 does not change the assembly curvature, shown in Fig. 4D. However, it dramatically alters the assembly pathway. Tetramers replace trimers as the most populated oligomeric intermediates, shown in Figs. 4F and G. Moreover, the assembled surfaces are filled with holes due to the incompatible symmetry of tetramers to tile the surface, shown in Fig. 4D. In addition, simulations with subunits in conformations based on hexameric template83 but with interactions modeled after the pentameric template81 rarely produced pentameric assemblies. It suggests that the protein backbone structure dominants the assembly curvature, while interactions play an assisting role. Taken together, their results imply the existence of dynamic heterogeneity at the two ends of the capsids to promote the HIV CAs to adopt pentamer compatible conformations for the sharp curvature formation. More refined CG models were applied to probe various oligomerization properties of the HIV CAs. Hicks and Henley extracted the elastic parameters of the 2D HIV CA sheet from pairwise all-atom MD simulations of HIV CAs102, which agrees with the value obtained by recent indentation experiments by AFM101. Yu and Hagan analyzed the water-density fluctuation at the CTDs of the HIV CAs and showed that the dimer interface stays wet as the proteins dimerize145, consistent with recent crystallography results29. Zhu and Chen established a residue-specific interaction potential for CG simulations of the HIV CAs146, which can be used to further enhance the resolution of CG simulations to the individual residue level. The third approach to probe the HIV CA assembly mechanism is to use rate equations to model assembly kinetics. Tsiang et al. proposed a simplified model to describe how individual CA monomers and dimers combine into hexameric sheets based on the structural models of the HIV CA hexameric and tubular assemblies80, 83. According to their oligomerization model, a set of rate equations were set up to describe the turbidity data of the HIV CA assembly141. Their results showed that the trimer of dimers are the rate-limited oligomeric intermediate. This agrees with the cryoEM identification of the trimer interface80, and the prediction from simulations with CG model that captures the HIV CA backbone structure8, 126, 142-144. In addition, trimers of dimeric HIV CAs were observed by AFM as building blocks to assemble into large structures on lipids by Miles and Frankel140. Recently, Sadre-Marandi et al. took an approach similar to that used by Endres and Zlotnick147 to analyze the assembly kinetics of HIV CAs at the initial nucleation stage, with elaborate numeration of all possible oligomerization pathways148. Their results confirmed the importance of 14 ACS Paragon Plus Environment

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trimers of dimeric subunits to nucleate the assembly. However, we note that their model was based on the data of mutant proteins with disrupted dimer interface, which could potentially take different assembly pathway from that of the WT HIV CAs. Summary statements By now, it is fairly certain that the HIV capsids consist of largely the hexameric CA assembly. The available structural data suggest the existence of pentamers as the structural defects to incur the sharp curvatures on the conical HIV capsid surface81, 103, 104, 124, as proposed by the fullerene motif6. However, the existence of other structural defects are not completely excluded, as shown by the observation of holes in cryoEM studies of authentic HIV capsids120, 124. It is possible a conclusive evidence will be revealed soon, with the rapid improvement of cryoEM techniques. Compared to the achievements to characterize the HIV CA assembly structures, our knowledge of the assembly kinetics and mechanism is still rather limited. Trimers of dimeric subunits are consistently suggested to be the nucleation oligomeric species on the assembly pathway8, 126, 140-144. Hypotheses were proposed to explain the million dollar questions regarding the assembly mechanism121, 122, 133, 134, 143, 144: the mechanism for polymorphism, size and curvature control of the HIV CA assembly. Their validity remains to be further tested by experimental observations. To advance our understanding of the assembly process, techniques with higher spatial and temporal resolutions are needed. Until then, it will be a blind-men’s game to figure out the elephant by the hands. AUTHOR INFORMATION Corresponding Author To whom correspondence should be addressed: Bo Chen, Tel: +1 407 823 4494, email: [email protected].

ACKNOWLEDGMENT The author acknowledges proof reading of the manuscript by Jaekyun Jeon and Tommy O. Boykin II.

FUNDING This work is supported by the AFOSR YIP award, funding number FA9550-13-1-0150.

ABBREVIATIONS AFM: Atomic Force Microscopy; AIDS: Acquired Immune Deficiency Syndrome; CA: capsid protein; CG: coarse grain; cryoEM: cryo electron microscopy; CTD: C-terminal domain; CypA: cyclophilin A; EM: electron microscopy; EIAV: Equine Infectious Anemia virus; FM: fluorescence microscopy; HIV: Human Immunodeficiency Virus; MA: matrix protein; MD: molecular dynamics; MHR: major homology region; NC: nucleocapsid protein; N-MLV: N-tropic murin leukemia virus; NTD: N-terminal domain; RNP: ribonucleoprotein complex; RSV: Rous Sarcoma Virus; ssNMR: solid state NMR; SAXS/WAXS small and wide angle X-ray scattering; WT: wild type. REFERENCES [1] Ambrose, Z., and Aiken, C. (2014) HIV-1 uncoating: connection to nuclear entry and regulation by host proteins, Virology 454, 371-379. [2] Bocanegra, R., Rodriguez-Huete, A., Fuertes, M. A., del Alamo, M., and Mateu, M. G. (2012) Molecular recognition in the human immunodeficiency virus capsid and antiviral design, Virus Res. 169, 388-410. [3] Campbell, E. M., and Hope, T. J. (2015) HIV-1 capsid: the multifaceted key player in HIV-1 infection, Nature Reviews Microbiology 13, 471483. [4] Le Sage, V., Mouland, A. J., and Valiente-Echeverria, F. (2014) Roles of HIV-1 capsid in viral replication and immune evasion, Virus Res. 193, 116-129. [5] Li, J., and Wang, W. (2015) Progress in the study of HIV capsid structure and drug discovery, Yao xue xue bao = Acta pharmaceutica Sinica 50, 1088-1095. [6] Ganser, B. K., Li, S., Klishko, V. Y., Finch, J. T., and Sundquist, W. I. (1999) Assembly and analysis of conical models for the HIV-1 core, Science 283, 80-83. [7] Briggs, J. A. G., Simon, M. N., Gross, I., Krausslich, H. G., Fuller, S. D., Vogt, V. M., and Johnson, M. C. (2004) The stoichiometry of Gag protein in HIV-1, Nature Structural & Molecular Biology 11, 672-675. [8] Zhao, G., Perilla, J. R., Yufenyuy, E. L., Meng, X., Chen, B., Ning, J., Ahn, J., Gronenborn, A. M., Schulten, K., Aiken, C., and Zhang, P. (2013) Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics, Nature 497, 643-646.

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(2015) Hierarchical multi-step organization during viral capsid assembly, Colloids and Surfaces B-Biointerfaces 136, 674-677. [129] Bocanegra, R., Alfonso, C., Rodriguez-Huete, A., Angel Fuertes, M., Jimenez, M., Rivas, G., and Mateu, M. G. (2013) Association Equilibrium of the HIV-1 Capsid Protein in a Crowded Medium Reveals that Hexamerization during Capsid Assembly Requires a Functional CDomain Dimerization Interface, Biophysical Journal 104, 884-893. [130] Barrera, F. N., del Alamo, M., Mateu, M. G., and Neira, J. L. (2008) Envelope lipids regulate the in vitro assembly of the HIV-1 capsid, Biophysical Journal 94, L8-L10. [131] Bruinsma, R. F. a. K., W.S. (2015) Physics of Viral Shells, Annu. Rev. Condens. Matter Phys. 6. [132] Lidmar, J., Mirny, L., and Nelson, D. R. (2003) Virus shapes and buckling transitions in spherical shells, Phys. Rev. E 68. [133] Nguyen, T. T., Bruinsma, R. F., and Gelbart, W. M. (2005) Elasticity theory and shape transitions of viral shells, Phys. Rev. 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[140] Miles, P., and Frankel, D. (2014) Lipid directed assembly of the HIV capsid protein, Soft Matter 10, 9562-9567. [141] Tsiang, M., Niedziela-Majka, A., Hung, M., Jin, D., Hu, E., Yant, S., Samuel, D., Liu, X., and Sakowicz, R. (2012) A Trimer of Dimers Is the Basic Building Block for Human Immunodeficiency Virus-1 Capsid Assembly, Biochemistry 51, 4416-4428. [142] Grime, J. M. A., and Voth, G. A. (2012) Early Stages of the HIV-1 Capsid Protein Lattice Formation, Biophysical Journal 103, 1774-1783. [143] Xin Qiao, J. J., Jeff Weber, Fangqiang Zhu, and Bo Chen. (2015) Mechanism of polymorphism and curvature of HIV capsid assemblies probed by 3D simulations with a novel coarse grain model, BBA - General Subjects 1850, 2353-2367. [144] Xin Qiao, J. J., Jeff Weber, Fangqiang Zhu, and Bo Chen. (2015) Construction of a novel coarse grain model for simulations of HIV capsid assembly to capture the backbone structure and inter-domain motions in solution, Data in Brief 5, 506-512. [145] Yu, N., and Hagan, M. F. (2012) Simulations of HIV Capsid Protein Dimerization Reveal the Effect of Chemistry and Topography on the Mechanism of Hydrophobic Protein Association, Biophysical Journal 103, 1363-1369. [146] Zhu, F., and Chen, B. (2015) Monte Carlo Simulations of HIV Capsid Protein Homodimer, Journal of chemical information and modeling 55, 1361-1368. [147] Ceres, P., and Zlotnick, A. (2002) Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids, Biochemistry 41, 11525-11531. [148] Sadre-Marandi, F., Liu, Y., Liu, J., Tavener, S., and Zou, X. (2015) Modeling HIV-1 viral capsid nucleation by dynamical systems, Mathematical Biosciences 270, 95-105.

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Figure 1. Full-length structure and dynamics of the wild-type HIV CA. A. The HIV CA monomer as defined in 3J3Q.pdb8. The NTD and CTD are colored in red and orange, respectively. All secondary structure components are labeled. B. The NTDs sam-ple a large conformation space around the rigid CTD dimer (white ribbons) in solution64. The blue and red transparent regions demonstrate a reweighted probability at 50% and 10% of the maximum. C. 2D contour projection of distribution of the posi-tion of the NTD centroid relative to CTD. D. The structures of the protein corresponding to the six labeled clusters are shown in ribbon diagram, with the NTD labeled in green and CTD in white. Figures B to D are reprinted with permission from ref. 64. Copyright (2013) American Chemical Society. 171x159mm (300 x 300 DPI)

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Figure 2. Structural models of various HIV CA assemblies. A. and B. The fullerene motif to create various shaped assemblies from p6 hexameric lattice with pentamer insertion. To create a cone from a p6 hexameric lattice, a continuous patch of hex-amers can be removed between the vertical and the other arrow-headed lines (P =1 to 5), and seal the remaining lattice togeth-er without disruption of the lattice, as shown at the top of A. By doing so, the tip angles of the cones created will only have five possible values: 112.9° of P=1, 83.6° of P=2, 60° of P=3, 38.9° of P=4, and 19.2° of P=5, as shown at the bottom of A. Then, ac-cording to Euler’s theorem, exactly 12 pentamers are also needed to close the cone, and their positions in the hexameric lattice will determine the assembly shape. A fullerene cone with 19.2° tip angle is shown on the left of B, with seven pentamers distrib-uted at the broad end and five pentamers at the narrow tip of the cone, resembling the typical authentic HIV capsid. Alterna-tively, a symmetric spherical assembly can be obtained if the pentamers are uniformly dispersed in the hexameric lattice, shown on the right of B. All pentamers in B are shaded. C. and D. The structural models of the WT HIV CA hexamer in hydrat-ed and dehydrated states, according to pdb 4XFX and 4XFY29, respectively. E. The structural model 3P05.pdb of an

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isolated pentamer formed by mutant HIV CAs with disrupted dimer interface81. Molecules are plotted in ribbon model, with each sub-unit in a different color. The dots in C and D are water molecules in the crystal. F. All-atom model of the HIV capsid generated combining cryoEM and MD simulation. All CTDs are colored in red. All NTDs in hexamers and pentamers are colored in blue and green, respectively. G. Interactions between a dimeric HIV CAs and the neighboring subunits in a flat 2D hexameric lattice. The hexameric assembly is based on 3H47.pdb83. The dimer interface between H9 is constructed based on 2KOD.pdb. The CTDs and NTDs of the dimer are plotted in orange and red, respectively. The CTDs and NTDs of their neighbors are plotted in green and cyan for distinction. The NTD-NTD, NTD-CTD, and the trimeric interfaces are shown in zoom-in views in indicated regions from H to J. The closest helices at each interface are identified, with solid lines connecting the points of closest dis-tances between their symmetric axes. Figures A and B are from ref. 6. Reprinted with permission from AAAS. Copyright (1999) AAAS. Figure F is adapted with permission from ref. 8. Copyright (2013) Nature Publishing Group. Figures G to J are adapted from ref. 143. Copyright (2015) Elsevier Ltd. 257x335mm (300 x 300 DPI)

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Figure 3. CryoEM images of authentic HIV CAs. A. and B. Tomographic slice through a viral particle exhibiting curling sheet and a CA with an extra sheet to its left side, respectively. C. Simulated structure of curling sheet in a roll. D. TEM images show-ing normal virions and abnormally large virions with free floating multi-CAs. The black scale bar is 500 nm. E. 3D rendering of tomograms of a typical multi-CA virions. Figure A-C are adapted with permission from ref. 120. Copyright (2013) Elsevier Ltd. Figure D and E are adapted with permission from ref. 124. Copyright (2015) Nature Publishing Group. 214x51mm (250 x 250 DPI)

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Figure 4. 3D CG simulations of HIV CA assemblies by the cylindrical model. A. The cylindrical model of the HIV CA based on 3J3Q.pdb8, capturing accurately the protein backbone structure shown in Fig. 1A. B. A curved hexameric lattice with 65.2 nm diameter assembled by a system of 128 dimeric subunits with variable NTDs to CTDs orientations derived from all-atom MD simulations. C. A sharp curvature assembly with 36.5 nm diameter formed by quasi-equivalent hexamer and pentamers, high-lighted in blue polygons. The simulation system consists of 128 dimeric subunits based on 3P05.pdb81 (pentamer structure) and 2KOD.pdb8 (44° crossing angle of H9 at dimer interface). D. A sharp curvature assembly with 40 nm diameter formed by te-tramers and a pentamer, highlighted in blue polygons. The simulation system consists of 128 dimeric subunits based on 3P05.pdb81 (pentamer structure) and the dimer interface modeled based on chains 7 and S in 3J34.pdb64 (34° crossing angle of H9 at dimer interface). E. to G. are the assembly pathways derived by the average of ten simulations with identical setup as in B to D, respectively. The dimeric subunits used in the simulations are shown in the inset of the figures. NTDs and CTDs are col-ored in red and orange, respectively. Figures adapted from ref. 143. Copyright (2015) Elsevier Ltd. 108x57mm (300 x 300 DPI)

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