Quantification of Entry Phenotypes of Macrophage-Tropic HIV-1 across a Wide Range of CD4 Densities

Joseph, Sarah; Arrildt, Kathryn T.; Swanstrom, Adrienne E.; Schnell, Gretja; Lee, Benhur; Hoxie, James A.; Swanstrom, Ronald

doi:10.1128/jvi.02477-13

Cited by 102 publications

(161 citation statements)

References 68 publications

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“…It is tempting to speculate that HIV-1 will reach maximum infectivity near to its natural number of spikes of 7 to 14 spikes per virion and that a dense array of Env is unnecessary for maximum infectivity. A caveat, however, is that infectivity requirements can differ with the type of target cell, particularly with cells having low levels of CD4 (e.g., macrophages) (63), or in the case of Envs of low intrinsic infectivity (48,50,51). Additional studies to determine the infectivity of hVLPs in which target cells and envelopes are varied will therefore be informative.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

Dense Array of Spikes on HIV-1 Virion Particles

Stano

Leaman

Kim

et al. 2017

J Virol

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“…The dependence on receptor level for viral entry can now be demonstrated most convincingly using the Affinofile cell line, in which the surface density of CD4 and/or CCR5 can be experimentally manipulated (21). Using this approach, it has been possible to identify M-tropic viruses, most often isolated from the cerebrospinal fluid (CSF) of subjects late in disease, by their ability to efficiently enter cells with low CD4 densities (CD4 low cells) as a surrogate marker for macrophage tropism (14,22). Although it is possible to observe differences in entry phenotype using MDMs, it is difficult to account for the inherent variability among both the viruses and the cells in unequivocally assigning cellular tropism.…”

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confidence: 99%

“…Differences in how macrophage tropism is defined can lead to substantially different observations about M-tropic viruses. Although it is widely observed that M-tropic viruses are able to enter more efficiently at low CD4 densities (14,19,(22)(23)(24)(25)(26) and are more sensitive to neutralization by soluble CD4 (sCD4) (27)(28)(29) than T-tropic viruses, few other characteristics are widely agreed upon. Similarly, several amino acid changes in the HIV-1 Env protein have been associated with macrophage tropism (24,25,27,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39), but the changes are not consistent across different subjects when macrophage tropism is defined as a distinct set of evolutionary variants within that subject (40).…”

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confidence: 99%

“…However, different preparations of MDMs can vary widely in their capacity to be infected-varying both between different donors and from the same donor at different times (13,14). Because MDMs have a lower surface density of CD4 than CD4 ϩ T cells, which is a significant impediment to entry by T-tropic viruses (14,18,19), it has been possible to use entry efficiency as a function of CD4 density to identify viruses that have adapted to entering macrophages. Initially, this was done using cells engineered to have either high or low levels of CD4 (20).…”

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confidence: 99%

“…These CXCR4-using T cell-tropic (X4 T-tropic) viruses use CXCR4 to target CD4 ϩ naive T cells (10,11), which express lower densities of CCR5 and higher densities of CXCR4 than do CD4 ϩ memory T cells (12,13). Alternatively, viral populations can evolve to use lower densities of the CD4 receptor, enabling more-efficient entry into macrophages, which express CD4 at densities 20-fold less than is found on CD4 ϩ memory T cells but express similar levels of the CCR5 coreceptor (14). Other studies have also observed that macrophages express lower levels of CD4 than CD4 ϩ T cells (13,15).…”

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confidence: 99%

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Phenotypic Correlates of HIV-1 Macrophage Tropism

Arrildt

LaBranche

Joseph

et al. 2015

J Virol

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HIV-1 is typically CCR5 using (R5) and T cell tropic (T-tropic), targeting memory CD4؉ T cells throughout acute and chronic infections. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize the phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M-and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation. IMPORTANCE HIV-1 typically replicates in CD4؉ T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses evolve and differ from CCR5-using T cell-tropic viruses may provide insights into viral evolution and pathogenesis within the central nervous system. We characterized the HIV-1 env viral entry gene from subject-matched macrophage-tropic and T cell-tropic viruses to identify entry features of macrophage-tropic viruses. We observed several differences between T cell-tropic and macrophage-tropic Env proteins, including functional differences with host CD4 receptor engagement and possible changes in the CD4 binding site and V1/V2 region. We also identified viruses with phenotypes between that of "true" macrophage-tropic and T cell-tropic viruses, which may represent evolutionary intermediates in a multistep process to macrophage tropism. HIV-1 host cell entry is determined solely by the virion surface protein Env. The Env protein precursor gp160 is cleaved into two proteins: the external gp120 protein and the membrane-spanning gp41 protein, which remain associated as a heterodimer and form trimers of these heterodimers. Attachment of gp120 to the host CD4 ...

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The evolution of HIV-1 entry phenotypes as a guide to changing target cells

Joseph

Swanstrom

2018

Journal of Leukocyte Biology

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Summary Sentence: The R5 T cell-tropic form of HIV-1 is the most abundant form and the evolutionary precursor to X4 T cell-tropic and macrophage-tropic variants. AbstractThrough a twist of fate the most common form of HIV-1, as defined by entry phenotype, was not appreciated until recently. The entry phenotype is closely linked to the target cell and thus to virus-host interactions and pathogenesis. The most abundant form of HIV-1 uses CCR5 as the coreceptor and requires a high density of CD4 for efficient entry, defining its target cell as the CD4+ memory T cell. This is the transmitted form of the virus, the form that is found in the blood, and the form that rebounds from the latent reservoir. When CD4+/CCR5+ T cells become limiting the virus evolves to use alternative target cells to support viral replication. In the CNS, the virus can evolve to use a cell that displays only a low density of CD4, while maintaining the use of CCR5 as the coreceptor. When this evolutionary variant evolves, it must be sustaining its replication in either macrophages or microglial cells, which display only a low density of CD4 relative to that on T cells. In the blood and lymphoid system, the major switch late in disease is from T cells expressing CD4 and CCR5 to T cells expressing CD4 and CXCR4, with a change in coreceptor specificity. Thus the virus responds in two different ways to different environments when its preferred target cell becomes limiting.

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Quantification of Entry Phenotypes of Macrophage-Tropic HIV-1 across a Wide Range of CD4 Densities

Cited by 102 publications

References 68 publications

Dense Array of Spikes on HIV-1 Virion Particles

Dense Array of Spikes on HIV-1 Virion Particles

Phenotypic Correlates of HIV-1 Macrophage Tropism

The evolution of HIV-1 entry phenotypes as a guide to changing target cells

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