Initiation of Infection

Catching HIV ‘in the act’ with 3Delectron microscopyLesley A. Earl1, Jeffrey D. Lifson2, and Sriram Subramaniam1

1 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD

20892, USA2 AIDS and Cancer Virus Program, SAIC-Frederick, Inc., Frederick National Laboratory, Frederick, MD 21702, USA

Review

The development of a safe, effective vaccine to preventHIV infection is a key step for controlling the disease on aglobal scale. However, many aspects of HIV biologymake vaccine design problematic, including the se-quence diversity and structural variability of the surfaceenvelope glycoproteins and the poor accessibility ofneutralization-sensitive epitopes on the virus. In thisreview, we discuss recent progress in understandingHIV in a structural context using emerging tools in 3Delectron microscopy, and outline how some of theseadvances could be important for a better understandingof mechanisms of viral entry and for vaccine design.

HIV infectionHIV infection is a major health issue worldwide, with morethan 34 million people currently living with HIV/AIDS(http://www.who.int). Despite decades of intensive re-search, a vaccine that can prevent transmission of thevirus remains elusive. HIV presents unique challengesto the design of effective vaccines: the rapid mutation rateof the virus generates abundant variants in response toselective pressures, resulting in extensive sequence andstructural variation. These characteristics enable the virusto escape the immune system in infected individuals.Further, they complicate the process of eliciting antibodiesthat can provide protection from the extraordinarily broadspectrum of antigenic variety displayed by the diversestrains of circulating HIV. Rational immunogen designin the face of this diversity is best served by a fundamentalunderstanding of essential features of both envelope gly-coprotein structure and function and mechanisms under-lying the entry of HIV into target cells.

HIV is an enveloped virus. A lipid bilayer membraneenvelope, acquired from the cellular membrane during theprocess of budding from an infected cell, surrounds theinternal nucleocapsid, which contains two copies of the viralgenome. Embedded in the envelope membrane and dis-played on the surface of the virion are envelope glycoprotein(Env) spikes [1]. Each individual Env spike is composed of atrimer of heterodimers of the transmembrane envelope

0966-842X/$ – see front matter .

Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tim.2013.06.004

Corresponding author: Subramaniam, S. (ss1@nih.gov).Keywords: cryo-electron tomography; focused ion beam scanning electron microscopy(FIB-SEM); ion abrasion scanning electron microscopy (IA-SEM); vaccine design;virus–cell interaction.

glycoprotein gp41 and the surface envelope glycoproteingp120 [2], derived from a common gp160 precursor. Thisstructure mediates infection through a multistep process inwhich gp120 binding to the cellular receptor CD4, followedby binding to a chemokine receptor (typically CXCR4 orCCR5), results in conformational changes that expose afusion domain in gp41, leading to fusion of the viral andtarget cell membranes and internalization of the viral nu-cleocapsid [3]. The primary target cells for HIV are CD4+ Tcells; however, there are many other cell types that interactwith and may become infected by HIV, including macro-phages and dendritic cells [4]. In turn, these cells can harborHIV, sometimes for very long periods of time, and transmitthe virus to other cells.

In this review, we provide an overview of selectedexamples of recent progress using new approaches in 3Delectron microscopy to better understand structuralaspects of the biology of HIV infection. We address threeseparate topics, each with important implications for pre-venting the spread of HIV: mechanisms of antibody neu-tralization, transfer of virus at a virological synapse, andour current understanding of the interactions at the virus–cell contact zone.

Neutralizing antibodies and EnvThe identification of broadly neutralizing antibodies,meaning antibodies that can bind and block entry of alarge proportion of circulating HIV strains, has been animportant objective in the quest for a vaccine [5]. The firstsuch antibodies were discovered nearly two decades ago[6–8], but were thought to be extremely rare. In recentyears, however, new methods have determined that up to25% of HIV-infected patients may develop such antibodies[9–13], and have allowed the rapid discovery of a number ofhighly potent broadly neutralizing antibodies [14–17].Although the structures of fragments of gp120 monomersbound to many of these neutralizing antibodies have beendetermined by X-ray crystallography [18–21], we do not yethave atomic resolution structures for trimeric Env in eithernative or antibody-bound states [22]. However, significantinsights into Env structure are beginning to emerge fromstudies of the Env complex using cryo-electron microscopyand cryo-electron tomography [1,22–27]. Individual Envspikes can be visualized in tomograms (i.e., 3D volumes) ofintact viruses and visualized either in slices through the

Trends in Microbiology, August 2013, Vol. 21, No. 8 397

(A) (B) (C)

(D) (E)

(F)

TRENDS in Microbiology

Figure 1. Quaternary structure of trimeric HIV-1 spikes on intact viruses in complex with neutralizing antibodies. (A,B) Slices through tomograms of plunge-frozen simian

immunodeficiency virus (SIV) mneE11S [23] and HIV-1 BaL variants [25], respectively. Scale bar in (A) is 35 nm. (C) 3D tomographic map of unliganded native HIV-1 BaL [1].

(D) Schematic representation of a virus illustrating the possible heterogeneity of antibody and ligand binding. The envelope glycoprotein (Env) is either unliganded (red) or

ligand (yellow-green) and antibody (green-blue) bound. (E,F) 3D tomographic density maps allow the determination of the location and orientation of antibody binding on

native Env spikes. Env is bound to VRC01 antibody alone (E) or to VRC01 antibody and 17b antibody (F) [33]. Crystal coordinates for glycoprotein (gp) 120 (red), VRC01

antibody (blue), and 17b antibody (green) are fitted into the maps. Coordinates for VRC01 are from the gp120–VRC01 complex [Protein Data Bank (PDB) ID: 3NGB] and are

aligned to coordinates for gp120 and 17b from the gp120–CD4/17b complex (derived from PDB ID: 1GC1).

Review Trends in Microbiology August 2013, Vol. 21, No. 8

tomogram (Figure 1A,B) or as 3D density maps at resolu-tions of approximately 20 A (Figure 1C). These densitymaps are acquired by averaging thousands of tomogramsof individual spikes, each of which provide information onthe structure of the trimer. Tomograms of viruses com-plexed with neutralizing antibodies or other ligands pro-vide insights into the location of the bound antibody on thesurface of Env spikes in situ on virions (Figure 1D). Byfitting three copies of the crystal structures of relevantgp120–Fab complexes into these density maps, molecularmodels for trimeric Env bound to one or more neutralizingantibodies can be obtained (Figure 1E,F).

Structural and biochemical studies have revealed thatbroadly neutralizing antibodies can bind to the Env spikeat a variety of locations. Antibodies such as VRC01, b12,HJ16, NIH45-46, 12A12, and 3BNC117 bind to the CD4binding site [6,16,28,29]. Other broadly neutralizing anti-bodies such as PG9, PG16, PGT145, and CH04 are thought

398

to bind primarily to the variable V1/V2 loops of gp120 [30–32]. Some others such as the glycan-dependent antibodies2G12 or PGT121 appear to mainly interact with the V3variable loop [31], whereas 17b or the human antibodyfragment m36 target the co-receptor binding site on gp120[25,33]. Others, such as 2F5 [34], 4E10 [35], 10E8 [20], andZ13e1 [36], bind to the highly conserved membrane-proxi-mal external region (MPER) of gp41 [37,38]. Although thebinding location and clonal specificities of many of theseantibodies have been characterized in detail, many of theantibodies have unusual features, including high levels ofsomatic hypermutation. Development of an immunogenthat can elicit these types of broadly neutralizing antibo-dies in the context of a vaccine remains an unsolvedproblem [17].

How do these antibodies function to block viral entry?Cryo-electron tomographic studies are beginning to pro-vide clues to the mechanisms that may underlie the action

TRENDS in Microbiology

(A)

(B)

Figure 2. Deep, HIV-laden channels in infected macrophages. Focused ion beam

scanning electron microscopy (FIB-SEM) imaging established that these channels

are connected to deep reservoirs that are contiguous with the extracellular

medium, and not discrete intracellular compartments. (A) Transverse section of

HIV-1 BaL-infected monocyte-derived macrophages imaged with FIB-SEM. Scale

bar is approximately 100 nm. (B) 3D reconstruction of the HIV-1-infected

macrophage demonstrating deep channels connecting a reservoir of HIV-1

virions (red) with the extracellular milieu [49].

Review Trends in Microbiology August 2013, Vol. 21, No. 8

of these different antibodies. Structural studies on nativeEnv trimers show that a key step in the entry process,triggered by CD4 binding, is a dramatic opening of thequaternary conformation of Env, involving outward move-ment of the three gp120 protomers and exposure of buriedregions of gp41. We now know that binding by 17b or m36antibodies appears capable of triggering the open confor-mation [25,33]. Despite this, these antibodies are neutral-izing, presumably because binding of these antibodies atthe apex of the spike blocks cell–virus contact. By contrast,the CD4 binding site-specific mAb VRC01 locks trimericEnv in a closed conformation that is very similar in struc-ture to that of the native, unliganded Env trimer, prevent-ing binding by the co-receptor [33]. Antibodies that bind tothe variable loop regions may block entry by a combinationof these mechanisms. MPER antibodies, such as Z13e1,very likely recognize an intermediate structure that occursafter CD4 binding and spike opening, but before viralfusion is initiated [39], suggesting that these antibodiescould block fusion by preventing gp41 rearrangements nec-essary for membrane fusion, if the kinetic challenges inher-ent in such a mode of action can be overcome. Although manybroadly neutralizing antibodies can function to preventcellular entry by HIV in vitro, and in fact have been shownto prevent infection by passive infusion in non-human pri-mate studies [40–42], it is an open question whether suchantibodies can be generated through vaccination, and bemaintained at sufficient titer to block virus infection andspread, including from one cell to another [43].

Cell-to-cell transmission of HIVAlthough CD4+ T cells are the primary targets of HIVinfection, many other cell types, including long-lived cellslike macrophages and dendritic cells, can either becomeproductively infected by the virus or can take up andsequester infectious virus in a form that can be transmittedto other cells, despite not becoming productively infectedthemselves [44–48]. Viruses sequestered in these cells canevade detection by the immune system. Insights into themechanism of sequestration of virus and the distribution ofHIV in these infected cells has come from a 3D imagingtechnique called focused ion beam scanning electron mi-croscopy (FIB-SEM). In this method, a scanning electronbeam is used to image the surface of a macroscopic cell ortissue specimen block that is progressively abraded, layerby layer, by a focused ion beam that removes a thin layer ofmaterial (typically �10 nm) at the surface. Electron micro-scopic images of the surface that are collected as thespecimen is consumed by the progressive abrasion processcan be combined to generate a 3D representation of thespecimen, with resolution allowing visualization of ultra-structural features of a cell. 3D images of HIV-infectedcells obtained using this approach led to the surprisingdiscovery that HIV particles in infected macrophages canbe present in virion channels (Figure 2) formed frominvaginations of the plasma membrane. These channelsare not closed internal compartments as was supposedfrom earlier 2D images of cross-sections through infectedcells, but conduits, contiguous with the extracellular mi-lieu, that could allow rapid transport of sequestered HIV toand from the external medium [49].

Transmission of viruses from antigen presenting cellssuch as macrophages or dendritic cells to T cells can occurat cell–cell contact zones called virological synapses(reviewed in [50]) via a process that is more effective thaninfection by cell-free virus [51,52]. Macrophages, for exam-ple, can become productively infected by HIV, and due totheir longevity, can spread the infection to T cells throughdirect contact over extended periods [44,45,53,54]. Thevirion channels described above may protect virions fromantibody-mediated neutralization, while still allowinginfection of T cells at virological synapses [49].

Dendritic cells, unlike macrophages, do not appear tobecome productively infected by HIV; nevertheless, thecells can also harbor infectious virus that can be transmit-ted to T cells across a virological synapse [4,55,56]. HIV canbe taken up by dendritic cells by a number of differentreceptors, including the C-type lectin DC-SIGN [57,58].Virus taken up by dendritic cells can be transferred toCD4+ T cells via virological synapses in a process that

399

Review Trends in Microbiology August 2013, Vol. 21, No. 8

usurps the normal physiological formation of dendriticcell–T cell conjugates, which proceed to generate theimmunological synapses involved in antigen presentation[59]. Structural studies of the close cell–cell contactsbetween HIV-carrying dendritic cells and uninfected Tcells using the FIB-SEM method described above haveshown that the dendritic cells encase the T cells in mem-branous sheets, creating extensive areas of membranecontact between the two cells (Figure 3). Moreover, withinthese areas of contact, a high local concentration of HIVbecomes trapped between the dendritic cell and T cell [60].T cells then appear to extend membrane protrusions to-wards pockets of virions (Figure 4A,B); these membraneextensions suggest HIV may trigger a reaction by unin-fected T cells that could increase the likelihood of infection[61,62]. Interestingly, CD4-blocking antibody appears tonegate this effect, leaving the virions clustered next tothe dendritic cell and away from the T cell, even in a closecell–cell contact (Figure 4C) [61].

The significant differences between HIV infection bycell-free virus and at the virological synapse have clear

(A)

(B)

TRENDS in Microbiology

Figure 3. Visualization of dendritic cell–T cell contact in the context of cell–cell HIV

transmission, and illustration of the sheets of dendritic cell membrane that encase

the T cell. These sheets of dendritic cell membrane often appear in thin section

images as dendrites. (A) Artistic representation of an HIV-1 virological synapse

between a dendritic cell (white) and a T cell (pink). (B) 2D slice (left) and 3D

reconstruction (right) of a virological synapse between a dendritic cell (pink) and

three T cells (yellow) imaged by focused ion beam scanning electron microscopy

(FIB-SEM). What appear as dendrites in 2D are shown to be membrane sheets in

3D [61].

400

implications for vaccine design. Not only are viruses withinsynapses likely to be shielded from potentially neutralizingantibodies, but the local concentrations of viruses may bemuch higher in virological synapses than in the context offree diffusion (reviewed in [63]). If transmission of virusacross virological synapses is involved in the process ofviral infection and dissemination of infection in a new host,including this mode into our paradigm of rational vaccinedesign and development may be necessary.

Viral entryViral entry into cells is a multistep process (reviewed in[64]). As noted above, the initial stages of viral entryinvolve gp120 on Env trimers binding to the cellularreceptor CD4, an opening of the Env spike, followed bybinding of gp120 to a chemokine co-receptor, such asCXCR4 or CCR5. However, a number of studies haveindicated that binding of a single Env spike is not sufficientto allow viral entry: multiple Env spikes are necessary forinitiating viral fusion with the cell membrane [64]. Consis-tent with such a process, clustering of Env trimer spikes onthe surface of virions can be observed directly by electrontomography (Figure 5A–C). This finding is particularlystriking in light of the limited number of spikes presenton most HIV-1 virions [1]. Studies of the contact zonebetween HIV and T cells have shown that there are anaverage of five to six Env spikes at the interface between abound virion and a target cell. This structure has beendesignated as an ‘entry claw’, and is speculated to beinvolved in mediating membrane fusion and viral entry[65] (Figure 5D). The presence of the entry claw suggests amechanism by which neutralizing antibodies that fail tosaturate the surface of an HIV virion may still be able toinhibit virus–cell fusion: if one of the spikes in the entryclaw is blocked by a neutralizing antibody, it is possiblethat the claw will fail to initiate fusion [66]. Thus under-standing the local concentration of antibodies at the virus–cell interface in vivo, and how this concentration corre-sponds to saturation of Env spikes on the surface of livevirus, is key to optimizing the function of an antibody-based preventive or therapeutic drug.

SIV and SHIV versus HIVBecause HIV does not productively infect non-human pri-mate species used in experimental models, preclinicalevaluation of vaccine concepts and candidates in animalmodels typically entails two phases, and involves the use ofa simian immunodeficiency virus (SIV), one of a family ofHIV-related primate lentiviruses that infect and can causeAIDS in macaques [67]. In the initial proof of conceptphase, the ability of an SIV vaccine that embodies thedesired vaccine approach to protect against a pathogenicSIV challenge is evaluated. If protection can be confirmed,then the immunogenicity of the corresponding HIV-basedvaccine is typically evaluated prior to a commitment toclinical testing. Alternatively, for Env-based vaccines, HIVimmunogens can be used in macaques, and protectionassessed using chimeric viruses, designated simian humanimmunodeficiency viruses (SHIV), comprised of HIV enve-lope encoding sequences grafted onto an SIV backbone.However, many of the SHIV that have been used to date for

(C)

(A) (B)

TRENDS in Microbiology

Figure 4. 3D imaging shows interdigitation of dendritic cell and T cell membranes at the virological synapse. (A,B) A virological synapse between an HIV-1-pulsed dendritic

cell (pink) and an uninfected T cell (yellow) was imaged by focused ion beam scanning electron microscopy (FIB-SEM) and reconstructed in 3D. (B) The reconstruction

shows that T cell membrane protrusions extend towards HIV-1 virions (red) on the surface of the dendritic cell. (C) 2D section through a virological synapse between a

dendritic cell pulsed with HIV-1 and an uninfected T cell treated with anti-CD4 antibody [61]. In the presence of CD4 blocking antibodies, virions remain at the surface of the

dendritic cells and are prevented from touching the T cell, indicating that cellular receptors are required for virion transfer within the virological synapse.

Review Trends in Microbiology August 2013, Vol. 21, No. 8

such studies have proven to be rather easier to protectagainst than typical SIV isolates, and by inference, HIV,and may not mediate authentic pathogenesis. Such viruseshave been somewhat useful in studies where the endpointis prevention of acquisition of infection, and substantiallyless useful in studies involving modulation of pathogenesispost-acquisition as an endpoint (reviewed in [67]). Conse-quently, SIV virions have been characterized, using someof the cryo-electron microscopy and tomographyapproaches described above, to study SIV Env spikes onvirions in situ [23]. As is the case for many other aspects ofAIDS virus biology, SIV largely recapitulates the essentialfeatures of HIV infection, but there are subtle differences,especially in quaternary conformation changes in the entryspike [23], that must be borne in mind when using SIV as amodel for HIV.

Concluding remarks: perspectives for HIV vaccinesNumerous HIV vaccine candidates have been developedand evaluated over the past two decades, some of themrationally designed from our structural knowledge of HIV[68–71]. Nevertheless, no vaccine formulation has yetproven sufficiently safe and effective at preventing HIV

infection to warrant licensure. The Phase III vaccine trialthat has reported the greatest efficacy to date has been theRV144 ‘Thai Trial’, based on pox virus vector priming andrecombinant protein boosting, with an efficacy of 31% [70].The limited protection observed has been correlated withnon-neutralizing IgG antibody responses, directed againstthe gp120 V1/V2 domain [72], and although additionalstudies are in progress to attempt to identify other poten-tial correlates of protection, there is broad agreement thatmore efficacious vaccines will be required.

Dramatic progress in the number of broadly neutraliz-ing monoclonal antibodies available and in the structuraldescription of the way these antibodies interact with HIVenvelope glycoproteins has increased our understanding oftheir function and mechanism of action. However, we haveyet to bridge the gap between antigenicity and immunoge-nicity. We understand in detail the antigenic bindingdeterminants on HIV envelope glycoproteins for broadlyneutralizing antibodies that are developed through thecourse of HIV infection, but we do not yet understandhow to induce formation of such antibodies by immuniza-tion of uninfected persons. Moreover, understanding howthese antibodies act mechanistically to block infection by

401

(A)

(D)

(B) (C)

TRENDS in Microbiology

Figure 5. Visualization of the entry claw formed at the contact zone between HIV and CD4+ T cells. The entry claw is thought to represent the formation of a macromolecular

complex between HIV envelope glycoproteins (Env) and cellular receptors prior to fusion between viral and cellular membranes. (A–C) Transmission electron microscopic

images of HIV-1 virions in contact with a CD4+ T cell. The entry claw is shown in a cluster of lines of density between the virus and cell. (D) Artistic visualization of the entry

claw, illustrating the nature of the virus–cell contact and clustering of Env spikes on the surface of the virion.

Review Trends in Microbiology August 2013, Vol. 21, No. 8

cell-free virions and inhibit transmission of virus acrossvirological synapses may be key to maximizing the efficacyof potential vaccines. In both these areas, future studiesusing 3D electron microscopy techniques hold promise forproviding informative insights. For blockade of infection bycell-free virus, a better understanding of the structuralbasis of interactions of antibodies with different functionalstates of the envelope spike, achieved through the methodsdescribed here, may help guide design of improved vaccineimmunogens. Similarly, studies of the spatial architectureof virological synapses, in combination with functionalstudies, may help better define the challenges for anti-body-mediated blockade of cell-to-cell virus transmission.

AcknowledgmentsWe thank Donald Bliss for assistance with illustrations. This work wassupported by funds from the National Institutes of Health (NIH)Intramural AIDS Targeted Antiviral Program and the Center for CancerResearch at the National Cancer Institute (to S.S.) and National CancerInstitute, National Institutes of Health Contract HHSN261200800001E(to J.D.L.).

402

References1 Liu, J. et al. (2008) Molecular architecture of native HIV-1 gp120

trimers. Nature 455, 109–1132 Wyatt, R. and Sodroski, J. (1998) The HIV-1 envelope glycoproteins:

fusogens, antigens, and immunogens. Science 280, 1884–18883 Dalgleish, A.G. et al. (1984) The CD4 (T4) antigen is an essential

component of the receptor for the AIDS retrovirus. Nature 312, 763–767

4 Piguet, V. and Steinman, R.M. (2007) The interaction of HIVwith dendritic cells: outcomes and pathways. Trends Immunol. 28,503–510

5 Nabel, G.J. et al. (2011) Progress in the rational design of an AIDSvaccine. Philos. Trans. R. Soc. B 366, 2759–2765

6 Burton, D.R. et al. (1994) Efficient neutralization of primary isolates ofHIV-1 by a recombinant human monoclonal antibody. Science 266,1024–1027

7 Muster, T. et al. (1993) A conserved neutralizing epitope on gp41 ofhuman immunodeficiency virus type 1. J. Virol. 67, 6642–6647

8 Trkola, A. et al. (1995) Cross-clade neutralization of primary isolates ofhuman immunodeficiency virus type 1 by human monoclonalantibodies and tetrameric CD4-IgG. J. Virol. 69, 6609–6617

9 Dhillon, A.K. et al. (2007) Dissecting the neutralizing antibodyspecificities of broadly neutralizing sera from humanimmunodeficiency virus type 1-infected donors. J. Virol. 81, 6548–6562

Review Trends in Microbiology August 2013, Vol. 21, No. 8

10 Li, Y. et al. (2007) Broad HIV-1 neutralization mediated by CD4-binding site antibodies. Nat. Med. 13, 1032–1034

11 Sather, D.N. et al. (2009) Factors associated with the development ofcross-reactive neutralizing antibodies during humanimmunodeficiency virus type 1 infection. J. Virol. 83, 757–769

12 Doria-Rose, N.A. et al. (2009) Frequency and phenotype of humanimmunodeficiency virus envelope-specific B cells from patients withbroadly cross-neutralizing antibodies. J. Virol. 83, 188–199

13 Stamatatos, L. et al. (2009) Neutralizing antibodies generated duringnatural HIV-1 infection: good news for an HIV-1 vaccine? Nat. Med. 15,866–870

14 Simek, M.D. et al. (2009) Human immunodeficiency virus type 1 eliteneutralizers: individuals with broad and potent neutralizing activityidentified by using a high-throughput neutralization assay togetherwith an analytical selection algorithm. J. Virol. 83, 7337–7348

15 Scheid, J.F. et al. (2009) Broad diversity of neutralizing antibodiesisolated from memory B cells in HIV-infected individuals. Nature 458,636–640

16 Scheid, J.F. et al. (2011) Sequence and structural convergence of broadand potent HIV antibodies that mimic CD4 binding. Science 333, 1633–1637

17 Kwong, P.D. and Mascola, J.R. (2012) Human antibodies thatneutralize HIV-1: identification, structures, and B cell ontogenies.Immunity 37, 412–425

18 Zhou, T. et al. (2010) Structural basis for broad and potentneutralization of HIV-1 by antibody VRC01. Science 329, 811–817

19 McLellan, J.S. et al. (2011) Structure of HIV-1 gp120 V1/V2 domainwith broadly neutralizing antibody PG9. Nature 480, 336–343

20 Huang, J. et al. (2012) Broad and potent neutralization of HIV-1 by agp41-specific human antibody. Nature 491, 406–412

21 Pejchal, R. et al. (2010) Structure and function of broadly reactiveantibody PG16 reveal an H3 subdomain that mediates potentneutralization of HIV-1. Proc. Natl. Acad. Sci. U.S.A. 107, 11483–11488

22 Merk, A. and Subramaniam, S. (2013) HIV-1 envelope glycoproteinstructure. Curr. Opin. Struct. Biol. 23, 268–276

23 White, T.A. et al. (2010) Molecular architectures of trimeric SIV andHIV-1 envelope glycoproteins on intact viruses: strain-dependentvariation in quaternary structure. PLoS Pathog. 6, e1001249

24 Harris, A. et al. (2011) Trimeric HIV-1 glycoprotein gp140 immunogensand native HIV-1 envelope glycoproteins display the same closed andopen quaternary molecular architectures. Proc. Natl. Acad. Sci. U.S.A.108, 11440–11445

25 Meyerson, J.R. et al. (2013) Molecular structures of trimeric HIV-1 Envin complex with small antibody derivatives. Proc. Natl. Acad. Sci.U.S.A. 110, 513–518

26 Frank, G.A. et al. (2012) Computational separation of conformationalheterogeneity using cryo-electron tomography and 3D sub-volumeaveraging. J. Struct. Biol. 178, 165–176

27 Zhu, P. et al. (2008) Cryoelectron tomography of HIV-1 envelope spikes:further evidence for tripod-like legs. PLoS Pathog. 4, e1000203

28 Wu, X. et al. (2010) Rational design of envelope identifies broadlyneutralizing human monoclonal antibodies to HIV-1. Science 329,856–861

29 Corti, D. et al. (2010) Analysis of memory B cell responses and isolationof novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS ONE 5, e8805

30 Walker, L.M. et al. (2009) Broad and potent neutralizing antibodiesfrom an African donor reveal a new HIV-1 vaccine target. Science 326,285–289

31 Walker, L.M. et al. (2011) Broad neutralization coverage of HIV bymultiple highly potent antibodies. Nature 477, 466–470

32 Bonsignori, M. et al. (2011) Analysis of a clonal lineage of HIV-1envelope V2/V3 conformational epitope-specific broadly neutralizingantibodies and their inferred unmutated common ancestors. J. Virol.85, 9998–10009

33 Tran, E.E. et al. (2012) Structural mechanism of trimeric HIV-1envelope glycoprotein activation. PLoS Pathog. 8, e1002797

34 Ofek, G. et al. (2004) Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with itsgp41 epitope. J. Virol. 78, 10724–10737

35 Cardoso, R.M. et al. (2005) Broadly neutralizing anti-HIV antibody4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 22, 163–173

36 Pejchal, R. et al. (2009) A conformational switch in humanimmunodeficiency virus gp41 revealed by the structures ofoverlapping epitopes recognized by neutralizing antibodies. J. Virol.83, 8451–8462

37 Weissenhorn, W. et al. (1997) Atomic structure of the ectodomain fromHIV-1 gp41. Nature 387, 426–430

38 Buzon, V. et al. (2010) Crystal structure of HIV-1 gp41 including bothfusion peptide and membrane proximal external regions. PLoS Pathog.6, e1000880

39 Harris, A.K. et al. (2013) HIV-1 envelope glycoprotein trimers displayopen quaternary conformation when bound to the gp41 membrane-proximal external-region-directed broadly neutralizing antibodyZ13e1. J. Virol. 87, 7191–7196

40 Shibata, R. et al. (1999) Neutralizing antibody directed against theHIV-1 envelope glycoprotein can completely block HIV-1/SIV chimericvirus infections of macaque monkeys. Nat. Med. 5, 204–210

41 Mascola, J.R. et al. (2000) Protection of macaques against vaginaltransmission of a pathogenic HIV-1/SIV chimeric virus by passiveinfusion of neutralizing antibodies. Nat. Med. 6, 207–210

42 Hessell, A.J. et al. (2009) Broadly neutralizing human anti-HIVantibody 2G12 is effective in protection against mucosal SHIVchallenge even at low serum neutralizing titers. PLoS Pathog. 5,e1000433

43 Chen, B.K. (2012) T cell virological synapses and HIV-1 pathogenesis.Immunol. Res. 54, 133–139

44 Sharova, N. et al. (2005) Macrophages archive HIV-1 virions fordissemination in trans. EMBO J. 24, 2481–2489

45 Carr, J.M. et al. (1999) Rapid and efficient cell-to-cell transmissionof human immunodeficiency virus infection from monocyte-derivedmacrophages to peripheral blood lymphocytes. Virology 265,319–329

46 Cameron, P.U. et al. (1992) Dendritic cells exposed to humanimmunodeficiency virus type-1 transmit a vigorous cytopathicinfection to CD4+ T cells. Science 257, 383–387

47 Nath, A. et al. (1995) Infection of human fetal astrocytes with HIV-1:viral tropism and the role of cell to cell contact in viral transmission. J.Neuropathol. Exp. Neurol. 54, 320–330

48 Piguet, V. and Sattentau, Q. (2004) Dangerous liaisons at thevirological synapse. J. Clin. Invest. 114, 605–610

49 Bennett, A.E. et al. (2009) Ion-abrasion scanning electron microscopyreveals surface-connected tubular conduits in HIV-infectedmacrophages. PLoS Pathog. 5, e1000591

50 Mothes, W. et al. (2010) Virus cell-to-cell transmission. J. Virol. 84,8360–8368

51 Chen, P. et al. (2007) Predominant mode of human immunodeficiencyvirus transfer between T cells is mediated by sustained Env-dependentneutralization-resistant virological synapses. J. Virol. 81, 12582–12595

52 Dimitrov, D.S. et al. (1993) Quantitation of human immunodeficiencyvirus type 1 infection kinetics. J. Virol. 67, 2182–2190

53 Aquaro, S. et al. (2002) Long-term survival and virus production inhuman primary macrophages infected by human immunodeficiencyvirus. J. Med. Virol. 68, 479–488

54 Brown, A. et al. (2006) In vitro modeling of the HIV-macrophagereservoir. J. Leukoc. Biol. 80, 1127–1135

55 Lekkerkerker, A.N. et al. (2006) Viral piracy: HIV-1 targets dendriticcells for transmission. Curr. HIV Res. 4, 169–176

56 Wang, J.H. et al. (2007) Functionally distinct transmission of humanimmunodeficiency virus type 1 mediated by immature and maturedendritic cells. J. Virol. 81, 8933–8943

57 Arrighi, J.F. et al. (2004) DC-SIGN-mediated infectious synapseformation enhances X4 HIV-1 transmission from dendritic cells to Tcells. J. Exp. Med. 200, 1279–1288

58 Gringhuis, S.I. et al. (2010) HIV-1 exploits innate signaling by TLR8and DC-SIGN for productive infection of dendritic cells. Nat. Immunol.11, 419–426

59 Vasiliver-Shamis, G. et al. (2010) HIV-1 virological synapse is notsimply a copycat of the immunological synapse. Viruses 2, 1239–1260

60 McDonald, D. et al. (2003) Recruitment of HIV and its receptors todendritic cell-T cell junctions. Science 300, 1295–1297

61 Felts, R.L. et al. (2010) 3D visualization of HIV transfer at thevirological synapse between dendritic cells and T cells. Proc. Natl.Acad. Sci. U.S.A. 107, 13336–13341

403

Review Trends in Microbiology August 2013, Vol. 21, No. 8

62 Nikolic, D.S. et al. (2011) HIV-1 activates Cdc42 and inducesmembrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation. Blood 118, 4841–4852

63 Zhong, P. et al. (2013) Cell-to-cell transmission of viruses. Curr. Opin.Virol. 3, 44–50

64 Blumenthal, R. et al. (2012) HIV entry and envelope glycoprotein-mediated fusion. J. Biol. Chem. 287, 40841–40849

65 Sougrat, R. et al. (2007) Electron tomography of the contact between Tcells and SIV/HIV-1: implications for viral entry. PLoS Pathog. 3, e63

66 Dobrowsky, T.M. et al. (2010) Organization of cellular receptors into ananoscale junction during HIV-1 adhesion. PLoS Comput. Biol. 6,e1000855

67 Lifson, J.D. and Haigwood, N.L. (2012) Lessons in nonhuman primatemodels for AIDS vaccine research: from minefields to milestones. ColdSpring Harb. Perspect. Med. 2, a007310

404

68 Flynn, N.M. et al. (2005) Placebo-controlled phase 3 trial of arecombinant glycoprotein 120 vaccine to prevent HIV-1 infection.J. Infect. Dis. 191, 654–665

69 Pitisuttithum, P. et al. (2006) Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120HIV-1 vaccine among injection drug users in Bangkok, Thailand.J. Infect. Dis. 194, 1661–1671

70 Rerks-Ngarm, S. et al. (2009) Vaccination with ALVAC and AIDSVAX toprevent HIV-1 infection in Thailand. N. Engl. J. Med. 361, 2209–2220

71 O’Connell, R.J. et al. (2012) Human immunodeficiency virus vaccinetrials. Cold Spring Harb. Perspect. Med. 2, a007351

72 Karasavvas, N. et al. (2012) The Thai Phase III HIV Type 1 Vaccinetrial (RV144) regimen induces antibodies that target conservedregions within the V2 loop of gp120. AIDS Res. Hum. Retroviruses28, 1444–1457