Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

Postler, Thomas S.; Martinez-Navío, José M.; Yuste, Eloísa; Desrosiers, Ronald C.

doi:10.1128/jvi.06463-11

Cited by 19 publications

(25 citation statements)

References 66 publications

(77 reference statements)

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“…hVLPs also showed no binding with an antibody to the Kennedy epitope of the CTT, Chessie 8, in agreement with a prior report that this epitope is generally not exposed on virions ( Fig. 2B) (40,41).…”

supporting

confidence: 91%

Dense Array of Spikes on HIV-1 Virion Particles

Stano

Leaman

Kim

et al. 2017

J Virol

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HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e., ϳ7 to 14. This exceptional feature has been associated with avoidance of humoral immunity, i.e., B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however, these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence-activated cell sorting (FACS) to sort for cells that are recognized by trimer cross-reactive broadly neutralizing antibody (bnAb) and not by nonneutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions (P ϭ 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and to display more than a 10-fold increase in spikes per particle by electron microscopy (average, 127 spikes; range, 90 to 214 spikes). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of "cell factory" selection were required for the high-Env phenotype. hVLPs showed greater infectivity than standard pseudovirions but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env.IMPORTANCE The paucity of spikes on HIV is a unique feature that has been associated with evasion of the immune system, while increasing spike density has been a goal of vaccine design. Increasing the density of Env by modifying it in various ways has met with limited success. Here, we focused instead on the producer cell. Cells that stably express HIV spikes were screened on the basis of high binding by bnAbs and low binding by nonneutralizing antibodies. Levels of spikes on cells correlated well with those on progeny virions. Importantly, high-Env virus-like particles (hVLPs) were produced with a manifest array of well-defined spikes, and these were shown to be superior in activating desirable B cells. Our study describes HIV particles that are densely coated with functional spikes, which should facilitate the study of HIV spikes and their development as immunogens.KEYWORDS antigenicity, broadly neutralizing antibodies, envelope glycoprotein, fluorescence-activated cell sorting, HIV-1, vaccine design, virus-like particles, cellPACK, electron microscopy, viral infectivity H uman immunodeficiency virus type 1 (HIV-1) displays around 7 to 14 envelope (Env) spikes per virion (1-3). This low number of spikes is unusual for enveloped viruses in comparison to numbers for influenza virus (ϳ400 to 500 spikes), vesicular

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supporting

confidence: 91%

Dense Array of Spikes on HIV-1 Virion Particles

Stano

Leaman

Kim

et al. 2017

J Virol

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“…These cellular results are in contrast to a study using GFP-fused gp41 truncation mutants where gp41 CTT was only observed in a cytoplasmic orientation in Env-expressing cells [35]. More recently, Desrosiers and colleagues have observed apparent exposure of CTT sequences on the surface of Env-expressing cells [36]. However, their results suggested that apparent CTT exposure was due to “shedding” of Env from expressing cells that subsequently bound to the surface of non-expressing cells and not that the CTT was natively exposed [36].…”

Section: Introductioncontrasting

confidence: 80%

“…More recently, Desrosiers and colleagues have observed apparent exposure of CTT sequences on the surface of Env-expressing cells [36]. However, their results suggested that apparent CTT exposure was due to “shedding” of Env from expressing cells that subsequently bound to the surface of non-expressing cells and not that the CTT was natively exposed [36]. Attempts to interpret and condense the results of these studies into a topological model for the CTT are hindered by the limitations of the differing techniques used by each group to determine CTT topology.…”

Section: Introductionmentioning

confidence: 99%

Detailed Topology Mapping Reveals Substantial Exposure of the “Cytoplasmic” C-Terminal Tail (CTT) Sequences in HIV-1 Env Proteins at the Cell Surface

et al. 2013

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Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20–50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.

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“…By comparison, the existence of neutralizing antibodies directed against the so-called Kennedy epitope of HIV-1 has been used as an argument supporting a topology switch of the (much larger) gp41 cytosolic tail [18]–[20]. The same issue has additionally been discussed in connection with the cytosolic tail of SIV gp41 and put in relation with other known cases [21].…”

Section: Discussionmentioning

confidence: 99%

Multiple Antibody Targets on Herpes B Glycoproteins B and D Identified by Screening Sera of Infected Rhesus Macaques with Peptide Microarrays

et al. 2014

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Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.

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Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

Cited by 19 publications

References 66 publications

Dense Array of Spikes on HIV-1 Virion Particles

Dense Array of Spikes on HIV-1 Virion Particles

Detailed Topology Mapping Reveals Substantial Exposure of the “Cytoplasmic” C-Terminal Tail (CTT) Sequences in HIV-1 Env Proteins at the Cell Surface

Multiple Antibody Targets on Herpes B Glycoproteins B and D Identified by Screening Sera of Infected Rhesus Macaques with Peptide Microarrays

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