# Mechanisms of HIV-1 cell-to-cell transfer to myeloid cells

Mingyu Han, Marie Woottum, Rémi Mascarau, Zoï Vahlas, Christel Vérollet, Serge Benichou

► To cite this version:

Mingyu Han, Marie Woottum, Rémi Mascarau, Zoï Vahlas, Christel Vérollet, et al.. Mechanisms of HIV-1 cell-to-cell transfer to myeloid cells. Journal of Leukocyte Biology, 2022, Focus Issue: Targeted Science - Special Focus Issue, 112 (5), pp.1261-1271. 10.1002/jlb.4mr0322-737r. hal-03835443

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Submitted on 2 Nov 2022

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Distributed under a Creative Commons Attribution 4.0 International License
DOI: 10.1002/JLB.4MR0322-737R

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JLB
JOURNAL OF
LEUKOCYTE
BIOLOGY

MEETING REVIEW

# Mechanisms of HIV-1 cell-to-cell transfer to myeloid cells

Mingyu Han<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Marie Woottum<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Rémi Mascarau<fnref n="4" />,<fnref n="5" />,<fnref n="6" />

Zoï Vahlas<fnref n="4" />,<fnref n="5" />,<fnref n="6" />

Christel Verollet<fnref n="4" />,<fnref n="5" />,<fnref n="6" />

Serge Benichou<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

**Correspondence**

Serge Benichou, Institut Cochin, 22 rue Méchain, Paris 75014, France.

Email: [serge.benichou@inserm.fr](mailto:serge.benichou@inserm.fr)

**Summary sentence:** Review of the mechanisms involved in HIV-1 cell-to-cell transfer to myeloid cells.

**Abstract**

In addition to CD4+ T lymphocytes, cells of the myeloid lineage such as macrophages, dendritic cells (DCs), and osteoclasts (OCs) are emerging as important target cells for HIV-1, as they likely participate in all steps of pathogenesis, including sexual transmission and early virus dissemination in both lymphoid and nonlymphoid tissues where they can constitute persistent virus reservoirs. At least in vitro, these myeloid cells are poorly infected by cell-free viral particles. In contrast, intercellular virus transmission through direct cell-to-cell contacts may be a predominant mode of virus propagation in vivo leading to productive infection of these myeloid target cells. HIV-1 cell-to-cell transfer between CD4+ T cells mainly through the formation of the virologic synapse, or from infected macrophages or dendritic cells to CD4+ T cell targets, have been extensively described in vitro. Recent reports demonstrate that myeloid cells can be also productively infected through virus homotypic or heterotypic cell-to-cell transfer between macrophages or from virus-donor-infected CD4+ T cells, respectively. These modes of infection of myeloid target cells lead to very efficient spreading in these poorly susceptible cell types. Thus, the goal of this review is to give an overview of the different mechanisms reported in the literature for cell-to-cell transfer and spreading of HIV-1 in myeloid cells.

**KEYWORDS**

cell-cell fusion, cell-to-cell transfer, HIV-1, myeloid cells

# 1 | INTRODUCTION

HIV-1 targets and replicates in immune cells expressing its main entry receptor CD4, including CD4+ T lymphocytes but also cells of the myeloid lineage such as macrophages, dendritic cells (DCs), and osteoclasts (OCs).<fnref n="1" />⁻<fnref n="6" /> Macrophages and DCs have been proposed as the first cells targeted by HIV-1 after sexual transmission and are then involved in early virus dissemination to lymphoid organs.⁷,⁸ Macrophages then participate, in addition to latently infected CD4+ T cells, to the for-

mation of persistent virus reservoirs in numerous host tissues.⁹⁻¹² Virus spreading in OCs, myeloid cells specialized in bone resorption, may also contribute to the bone disorders observed in HIV-1-infected patients.<fnref n="6" />,¹³

In addition to CD4, virus entry in these HIV-1 target cells absolutely requires cell surface expression of the CCR5 and/or CXCR4 chemokine coreceptors that participate in virus entry through sequential interactions with the viral envelope gp120/gp41 glycoproteins present at the surface of the virus particles.¹⁴,¹⁵ The gp120 surface glycoprotein initially binds to CD4 and then to the chemokine receptor leading to conformational changes of the gp41 transmembrane glycoprotein that finally mediates fusion of the viral envelope with the target cell membrane. This process of virus entry is related to cell-free virus particle infection and is very efficient for in vitro infection of dividing CD4+

<footnote n="1">¹Institut Cochin, Inserm U1016, Paris, France</footnote>

<footnote n="2">²Centre National de la Recherche Scientifique
CNRS UMR8104, Paris, France</footnote>

<footnote n="3">³Faculty of Health, University of Paris Cité,
Paris, France</footnote>

<footnote n="4">⁴Institut de Pharmacologie et Biologie
Structurale, IPBS, Université de Toulouse,
CNRS, Toulouse, France</footnote>

<footnote n="5">⁵International Research Project (IRP) CNRS,
Toulouse, France</footnote>

<footnote n="6">⁶International Research Project (IRP), CNRS,
Buenos Aires, Argentina</footnote>

<footnote>**Abbreviations:** DC, dendritic cell; iDC, immature dendritic cell; OC, osteoclast; T/F, transmitted/founder; SAMHD1, SAM domain and HD domain-containing protein 1; APOBEC3, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3; TRIM, TRlpartite Motif protein; IFITM, InterFeron-Induced TransMembrane protein; CNS, central nervous system; MGCs, multinucleated giant cells; TNTs, tunneling nanotubes; Mtb, Mycobaterium tuberculosis; Vpu, viral protein U.</footnote>
human T-cell lines. In contrast, primary target cells, and especially non-cycling myeloid cell targets such as macrophages, are poorly infected, even in vitro, by cell-free viruses. The viral envelope also determines the cellular tropism of the HIV-1 strains that can be classified in 3 types: CXCR4-using T cell-tropic, CCR5-using T cell-tropic (also called non-macrophage-tropic), and macrophage-tropic strains using either CCR5 or CXCR4, or both, as coreceptors.¹⁶ Although CCR5-using viruses, including sexually transmitted/founder (T/F) viruses,¹⁷,¹⁸ predominate during early, acute, and asymptomatic phases of infection, CXCR4-using variants can emerge at later stage of infection and are mostly associated with advanced disease progression.¹⁹⁻²¹

The first block to efficient macrophage infection is related to virus entry and specific cellular tropism of the different HIV-1 variants found in infected individuals and characterized as non-M-tropic using cell-free in vitro infection assays. Poor virus replication in these myeloid cells is also largely related to the high expression and/or efficiency of host cell restriction factors that inhibit postentry replication steps of the viral life cycle, such as SAMHD1 (SAM domain and HD domain-containing protein 1), an enzyme that inhibits the reverse transcription process through depletion of the pool of intracellular nucleotides necessary for efficient synthesis of the viral DNA, but also members of the APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3), TRIM (TRIpartite Motif protein), and IFITM (InterFeron-Induced TransMembrane protein) families.²²

In addition to cell-free virus infection, there is substantial evidence indicating that cell-to-cell transfer of HIV-1 through direct contacts between infected virus-donor cells and recipient targets cells likely represents the dominant mode of virus dissemination in vivo and may allow for productive infection of both lymphoid and myeloid target cells. At least in vitro, this cell-to-cell mode of virus transfer is very efficient for virus dissemination in target cells,²³ such as myeloid cells,²⁴ poorly susceptible to cell-free virus infection but detected in vivo in many tissues, mainly in the central nervous system (CNS), and also in other tissues, including lymph nodes, spleen, lungs, and digestive and genital tracts.⁹⁻¹² Remarkably, these infected myeloid cells are very often found as multinucleated giant cells (MGCs) in tissues.⁶,⁹,¹¹,¹²,²⁵⁻³³ Although CD4+ T cells do not substantially contribute to infection of the CNS, both parenchymal microglia, the resident macrophage cell lineage of the brain, and infected perivascular monocytes/macrophages migrating through the blood-brain barrier are the main cells infected by HIV-1 in the brain. They participate in the development of HIV-associated neurocognitive disorders, which can persist in infected patients despite effective suppressive antiretroviral therapy.²⁵ Cell-to-cell virus dissemination may also enable virus to escape elimination by the immune system, including neutralizing antibodies, and antiretroviral drugs.³⁴⁻³⁹

HIV-1 cell-to-cell transfer between CD4+ T cells, mainly through the formation of the so-called virologic synapse,⁴⁰⁻⁴² or from infected macrophages or DCs to CD4+ T cell targets, have been extensively described in vitro.²³,⁴³⁻⁴⁹ In addition, recent reports demonstrate that myeloid cells can be also productively infected through virus cell-to-cell transfer for more efficient spreading in these poorly susceptible cell types. As myeloid cells are now emerging as important target

cells involved in all steps of HIV-1 pathogenesis and in viral persistence in tissues of infected individuals,¹⁰,⁵⁰,⁵¹ even under conditions of antiretroviral treatment, the goal of this review is to discuss the different mechanisms reported in the literature regarding HIV-1 cell-to-cell spread leading to productive infection of myeloid cells. The first part of the review will be related to the mechanisms of homotypic virus cell-to-cell transfer between macrophages, whereas the second part will focus on the 2 different mechanisms reported for heterotypic virus cell-to-cell dissemination from virus-donor-infected CD4+ T cells to myeloid cell targets, including macrophages, DCs, and OCs.

## 2 | HOMOTYPIC HIV-1 CELL-TO-CELL TRANSFER BETWEEN MACROPHAGES

### 2.1 | Virus transfer between myeloid cells through the formation of tunneling nanotubes

The role in intercellular communication of several types of membrane protrusions, including lamellipodia, pseudopodia, filopodia, as well as various other structures such as tunneling nanotubes, has been widely described both in vitro and in vivo as a major mechanism of transmission of information between different types of cells in many organs and tissues.⁵²⁻⁵⁷ Among these specialized structures, the formation of membrane nanotubes corresponds to protrusions that extend from the plasma membrane and enable cells to touch over long distances, sometimes over 100 µm, as reported between T cells⁵⁸ but also between myeloid cells, such as macrophages and DCs.⁵⁹⁻⁶³ These membrane tunneling protrusions are regrouped as 2 main types: close-ended nanotubes and open-ended nanotubes also known as tunneling nanotubes (TNTs).⁴⁹,⁵³,⁶¹,⁶⁴,⁶⁵ TNTs are dynamic structures that contain F-actin and do not touch the substrate, meaning that they are hovering above the substrate in the culture medium in contrast to other membrane protrusions such as filopodia, which are attached to the substrate. They are again subdivided in 2 classes depending of their thickness and cytoskeleton composition: thin TNTs (<0.7 µm) and thick TNTs (>0.7 µm) containing microtubules.⁵⁸,⁵⁹,⁶⁶⁻⁶⁸ Although TNTs are physiologically involved in multiple cell-to-cell transfer of many cellular components and macromolecules, and even big cellular organelles such as mitochondria, several studies have demonstrated that both thin and thick TNTs could also transport pathogens, such as bacteria or viruses, including HIV-1 particles leading to efficient virus cell-to-cell transfer between CD4+ T cells or macrophages (Figure 1(A)).⁵⁸,⁵⁹,⁶⁶,⁶⁸⁻⁷⁰ In CD4+ T cells, in vitro HIV-1 infection does not seem to trigger or increase the formation of TNTs, but viral particles can be quickly transferred from a CD4+ T cell to another through this intercellular connection.⁵⁸ In contrast, it was reported that the number of both thin and thick TNTs was increased in HIV-1-infected macrophages compared with uninfected cells.⁵⁹,⁶⁸ In an in vivo context using HIV-1-infected “humanized mice,” TNT-like structures containing the viral p24 capsid protein, as well as the viral mRNA and nonintegrated DNA have been documented in tissues of infected animals.⁷¹ TNT formation was increased in both macrophages and CD3+ lymphocytes early after
**FIGURE 1** Intercellular structures and processes involved in cell-to-cell transfer of HIV-1 in myeloid cells. Different pathways for HIV-1 cell-to-cell transfer between virus-donor-infected cells (in green) and target myeloid cells (in purple) have been described through (A) formation of tunneling nanotubes (TNTs), (B) homotypic cell-cell fusion, (C) phagocytosis of infected T cell, and (D) heterotypic cell-cell fusion

<figure><img src="image_3.png" /><figcaption>B Homotypic cell-cell fusion</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C Phagocytosis of infected T cell</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D Heterotypic cell-cell fusion</figcaption></figure>

<figure><img src="image_6.png" /></figure>

infection in lymph nodes, spleen, and lungs, whereas only macrophages were able to form TNTs in the brain. Interestingly, treatment of infected mice with the TAK779 CCR5 antagonist decreased TNT formation between CD3+ T cells in spleen and lungs, whereas it was enhanced between macrophages in lymph nodes and brain, suggesting that HIV-1 can continue to spread between macrophages in these tissues in a TNT-dependent manner despite antiretroviral therapy with TAK779.

Thin TNTs would be directly involved in the transport of virus particles,⁵⁹ whereas thick TNTs could transport HIV-1 constituents such as viral Gag and Env proteins through intracellular vesicles derived from cellular organelles like the endoplasmic reticulum and the Golgi network or from virus-containing compartments.⁵⁹,⁷⁰ These initial results were then confirmed by other studies describing the presence of these viral constituents into endocytic compartments that might allow efficient viral cell-to-cell transfer through TNTs formed between macrophages.⁷²,⁷³ In addition, the HIV-1-dependent formation of TNTs between adjacent macrophages likely favors subsequent cell-cell fusion for more efficient spreading in this target cell-type through formation of MGCs.⁶⁸,⁷⁴,⁷⁵

It was reported that the stimulation of TNT formation observed in HIV-1-infected macrophages is dependent and modulated by the viral Nef auxiliary protein,⁶⁶,⁷⁶ as HIV-1-induced TNT formation and cell-cell fusion leading to MGC formation are almost abolished when macrophages are infected by Nef-deficient viruses.⁶⁶,⁷⁴,⁷⁵ This role of Nef on TNT formation and cell-cell fusion is directly related to the capacity of Nef to modulate cytoskeletal dynamics,⁶,⁴⁹,⁷⁵,⁷⁷⁻⁷⁹ Other authors reported that Nef is also able to interact with members of the exocyst complex involved in membrane vesicle trafficking through the M-sec cellular protein.⁶⁶,⁷⁶,⁸⁰,⁸¹ This cytosolic protein, expressed in cells of the monocytic lineage, including macrophages, participates in the formation of TNTs.⁴⁹,⁸²,⁸³

Interestingly, tuberculosis is a risk factor in HIV-1-infected individuals, and we have revealed that the formation of TNTs was facilitated in HIV-1-infected macrophages in the context of coinfection with *Mycobacterium tuberculosis* (Mtb), the pathogen causing tuberculosis.

In vitro, many studies showed that tuberculosis environment favors HIV-1 replication in macrophages.⁶⁸,⁶⁹,⁸⁴⁻⁸⁶ Mtb infection induces a proinflammatory environment through the production of TFNα, IL-6, IL-1β, and IL-10 cytokines by HIV-1-infected macrophages that increases NFκB binding to the viral promotor initiating viral transcription in coinfected macrophages.⁶⁸,⁸⁵,⁸⁷⁻⁹² We have also reported that Mtb-associated microenvironments specifically induce formation of both thin and thick TNTs through an IL-10/STAT3 signaling pathway in HIV-1-infected macrophages.⁶⁸ Interestingly, the formation of TNTs induced by Mtb in HIV-1-infected macrophages is also dependent of Siglec-1, a receptor mainly expressed by myeloid cells, which has been involved in capture of several pathogens, including HIV-1, by macrophages.⁶⁹,⁹³,⁹⁴ Exposure of macrophages to Mtb-derived environment results in induction of type 1 IFN-I production leading to up-regulation of Siglec-1 expression on TNTs, which favors their stabilization for a more efficient cell-to-cell viral transfer between macrophages.⁶⁹

As mentioned above, the formation of these TNTs could also drive HIV-1 cell-to-cell transfer by favoring direct intercellular contacts between macrophages leading to cell-cell fusion and formation of infected MGCs.⁵⁹,⁶⁶,⁷⁴,⁷⁵

## 2.2 | Virus transfer between macrophages through homotypic cell-cell fusion

When infected by HIV-1, CD4+ T cells and macrophages express the viral envelope glycoproteins (gp120 and gp41) at the cell surface, and the fusogenic capacity of the viral envelope can induce fusion between infected donor cells and neighboring target cells expressing the CD4 receptor and the CCR5 and/or CXCR4 coreceptors to form infected syncytia (or MGCs).⁹⁵ Although the process of syncytia formation between infected CD4+ T cells has been largely documented in vitro but discussed for its in vivo relevance,²³ some recent reports indicate that this process of cell-cell fusion could be more relevant
for HIV-1 cell-to-cell infection of myeloid cells (Figure 1(B)). Infected multinucleated macrophages can be found in vivo in tissues of HIV-1-infected patients and nonhuman primates experimentally infected with the SIV, mainly in the CNS, but also in other lymphoid or nonlymphoid tissues.²⁷,²⁹,³¹,⁹⁶⁻⁹⁸ In the CNS of HIV-infected patients, the detection of MGCs has even been proposed by some authors as a hallmark of AIDS-associated neurologic disorders that can be observed even in patients under suppressive antiretroviral therapy.²⁵,²⁷,²⁹,³³,⁹⁶,⁹⁸⁻¹⁰²

In vitro, homotypic cell-cell fusion of HIV-1-infected myeloid cells has been described more specifically between macrophages, but also between OCs,⁶,⁷,⁰,⁷⁴,⁷⁵,¹⁰³ leading to the formation of long-lived infected MGCs that can produce high levels of infectious viral particles. This homotypic cell-cell fusion process is directly related to interactions between the viral envelope glycoproteins expressed at the surface of the infected virus-donor cell and the CD4 and chemokine coreceptors expressed at the surface of the target cell, but other molecular mechanisms linked to cytoskeleton reorganization could also participate in the modulation of this complex process.⁷⁰,⁷⁴,⁷⁵ The intrinsic propensity of cells of the myeloid lineage to mediate homotypic cell-cell fusion to form multinucleated myeloid cells both in physiologic and pathologic conditions could also facilitate HIV-1-mediated homotypic cell-cell fusion of myeloid cells through engagement of signaling pathways and effectors involved in programming of myeloid cells into a fusion-competent state depending of the cytokine environment.¹⁰⁴,¹⁰⁵ Although the formation in bones of functional multinucleated OCs is mainly orchestrated by the receptor activator of NF-κB ligand cytokine, alternative activation of macrophages by IL-4 and IL-13 can also promote homotypic macrophage cell-cell fusion and MGC formation.¹⁰⁵ The formation of these particular MGCs, also called foreign body giant cells, formed in response to organic and inorganic materials, has been studied at the molecular level,⁸² but the contribution of the cellular pathways mobilized by these cytokines in the cell-cell fusion of macrophages, OCs, and DCs observed during HIV-1 infection has not been yet investigated.

Again, it was reported that the viral Nef protein was involved in the modulation of the process to stimulate cell-cell fusion, as MGC formation was significantly decreased when macrophages are infected with Nef-deficient viruses compared with wild-type HIV-1. Furthermore, expression of the Nef protein was sufficient to trigger the formation of MGCs in the macrophage-like RAW264.7 cell line.⁷⁴ This mechanism of HIV-1 cell-cell fusion is related to the activation by Nef of Hck, a Src-family tyrosine kinase specifically expressed in phagocytes, through direct interaction between the polyproline motif of Nef and the SH3 domain of Hck.¹⁰⁶⁻¹⁰⁸ Even if the 2 p61 and p59 isoforms of Hck are activated by Nef, only activation of the lysosomal p61Hck isoform seems to trigger the formation of MGCs through its catalytic activity and its association with lysosomes containing several proteins including proteases that participate in Nef-induced cell-cell fusion between macrophages and MGC formation.⁷⁴ Despite their large size, these newly formed infected MGCs migrate faster in dense matrices (i.e., mesenchymal mode of migration)¹⁰⁹ than their mononucleated counterparts, again by a Nef-dependent mechanism. This effect of Nef on HIV-1-infected macrophage migration ability has been evi-

denced both in vitro and in vivo in transgenic mice expressing Nef.⁷⁴,⁷⁵ Thus, cell-cell fusion of infected macrophages and MGC formation may represent in vivo a very efficient route for viral dissemination and formation of resident HIV-1 reservoirs in different tissues such as the CNS.

## 3 | HETEROTYPIC HIV-1 CELL-TO-CELL TRANSFER FROM INFECTED CD4 T CELLS TO MYELOID TARGET CELLS

### 3.1 | Virus transfer in macrophages through phagocytosis of HIV-1-infected CD4+ T cells

As innate immune sentinels, one of the biologic function of macrophages is their capacity to detect and eliminate dying cells and infected cells through phagocytosis, a cellular process by which large particles (≥0.5 µm) are engulfed at the plasma membrane.²⁴,¹¹⁰,¹¹¹ In addition to this function, the team of Quentin Sattentau has proposed a new mechanism for HIV-1 cell-to-cell transfer from infected CD4+ T cells to macrophages through selective capture and engulfment of HIV-1-infected CD4+ T cells (Figure 1(C)), leading to infection of macrophages.¹¹² This in vitro observation may correlate with results obtained in SIV-infected macaques showing that myeloid cells from lymphoid tissues contain T cell markers and viral DNA originating from infected CD4+ T cells,¹¹³,¹¹⁴ although it is difficult to reconcile this finding with the phagocytosis process as it would involve a progressive degradation of the T-cell material.¹¹⁵

As expected, this engulfment (or phagocytosis) process is more pronounced and efficient when infected T cells are dead or dying.¹¹² Only CD4+ T cells infected by CCR5-using macrophage-tropic strains and in a lesser extend CCR5-using T/F viruses, which poorly infect macrophages in cell-free infection assays,¹¹⁶,¹¹⁷ led to productive infection of the target macrophages.¹¹² By contrast, no productive infection of the macrophage targets is observed after engulfment of T cells infected with non-macrophage-tropic CXCR4-using viruses, although these infected T cells are efficiently engulfed by target macrophages. This mechanism of engulfment of infected T cells is independent of the interactions between the viral envelope glycoproteins and cellular receptors (i.e., CD4 and CCR5/CXCR4) required for HIV-1 entry, as T cells infected with either wild-type or envelope-deficient viruses were equally captured by macrophages. Similarly, T cells infected with the laboratory-adapted lymphotropic CXCR4-using strain NL4.3 were efficiently captured. However, the subsequent step leading to productive infection of the macrophage target relies on interactions of the viral envelope with the CD4 receptor and the CCR5 coreceptor, as it is inhibited by antibodies or drugs targeting Env and CD4, and to a lesser extent by antagonists of CCR5, suggesting that this second step likely results from fusion between the viral envelope and the cellular membrane of the phagolysosomes.

Together, these results indicate that this process of macrophage infection through engulfment of infected CD4+ T cells is related to a nonconventional mechanism requiring further investigations to
understand why engulfment of infected T cells infected by CCR5-using macrophage-tropic and weakly macrophage-tropic T/F viruses lead to the productive infection of the macrophage targets. In contrast, CD4+ T cells infected by nonmacrophage-tropic CXCR4-using viruses are efficiently engulfed but failed to result in productive infection of macrophages targets. Interestingly, it was recently reported that the viral protein U (Vpu), a membrane-associated HIV-1 auxiliary protein that assists viral dissemination, can promote the susceptibility of macrophages to infection through phagocytosis of infected CD4+ T cells.<sup>118</sup> This role is related to the function of Vpu in the cell surface remodeling observed in HIV-1-infected cells through down-regulation of cell surface proteins such as CD4 and the antiviral host restriction factor BST2,<sup>119-122</sup> but also CD47.<sup>123,124</sup> It was shown that CD47 serves as a ligand of the inhibitory receptor signal regulatory protein-alpha (SIRPα) expressed on myeloid cells, and acts as a "don't eat me" signal.<sup>125</sup> By inducing down-regulation of CD47 from the surface of infected CD4+ T cells, Vpu thus promotes capture and phagocytosis of infected T cells by macrophages leading to productive infection of these target cells by a prototypic CCR5-using macrophage-tropic virus, but also by a T/F virus, which was not previously defined as macrophage tropic.<sup>118</sup>

## 3.2 | Virus transfer in macrophages through heterotypic cell-cell fusion with infected CD4+ T cells

Finally, recent studies from our team have revealed a new route for rapid and massive cell-to-cell transfer and dissemination of HIV-1 from infected CD4+ T cells toward the main target myeloid cells, including macrophages, but also OCs and immature DCs (iDCs).<sup>6,126,127</sup> This process of virus cell-to-cell transfer is completed by a 2-step cell-cell fusion mechanism leading to the productive infection of the myeloid target cells. In the first step, infected T cells establish rapid contacts with macrophages, OCs, or iDCs, resulting in cell-cell fusion with these target myeloid cells (Figure 1(D)). The newly formed Gag+ fused cells then acquire the ability to fuse with surrounding noninfected macrophages, iDCs, or OCs, leading to the formation of infected MGCs containing at least 1 nucleus coming from the initial fusion of infected T cells. These MGCs are able to produce high levels of fully infectious new virus particles. As already mentioned, infected macrophage-derived MGCs, but also DC-derived MGCs, can be found in vivo in tissues of HIV-1-infected patients. In the CNS of HIV-1-infected patients and SIV-infected monkeys, infected macrophages are very often detected as multinucleated cells.<sup>25,27,29,33,96,98,100,101</sup> The presence of HIV-1-infected multinucleated cells derived from myeloid cells was also found at the surface of the nasopharyngeal tonsils, adenoids, and parotid glands of HIV-1-infected patients.<sup>28,33,128,129</sup> This heterotypic and 2-step cell-cell fusion process described in vitro for virus transfer in myeloid cells agrees with the in vivo observations already mentioned showing that T cell markers are detected in myeloid cells from lymphoid tissues of SIV-infected macaques.<sup>113,114</sup>

Initially revealed using HIV-1 strains expressing CCR5-using macrophage-tropic viral envelopes, we recently obtained results suggesting that primary viral variants isolated from infected patients, using either CCR5 and/or CXCR4 and characterized by cell-free infection assays as non-macrophage tropic, including some R5-tropic T/F viruses, could be efficiently transferred from infected T cells and then disseminate in macrophages by cell-cell fusion for virus spreading through the formation of productively infected MGCs (SB and MH, results referenced in bioRxiv<sup>130</sup>). Although the viral tropism of the virus strains, defined by the coreceptor usage, is maintained between cell-free and cell-to-cell infection processes, the initial block of virus entry for efficient replication in macrophages could be overcome in T cell-to-macrophage cell-cell fusion, indicating that HIV-1 has a more prevalent tropism for macrophages than initially suggested. In agreement, a recent report shows that non-macrophage-tropic primary HIV-1 isolates can efficiently infect alveolar macrophages through contact with infected CD4+ T cells.<sup>131</sup> These findings indicate that the cellular tropism of primary HIV-1 isolates needs to be revisited to reflect the capacity of both CCR5- and CXCR4-using non-M-tropic variants, including some T/F viruses, to spread efficiently in myeloid cells via cell-to-cell transfer from infected T cells by cell-cell fusion. Compared with cell-free infection, our results show an enhancement of interactions of CCR5-using macrophage- and non-macrophage-tropic viral envelope glycoproteins with CD4 and CCR5 during T-cell/macrophage contacts. Enhanced envelope/receptor interactions may increase the capacity of HIV-1 envelopes, and particularly non-M-tropic envelopes, to trigger cell-cell fusion of infected T cells with macrophages. This enhancement results in a reduced dependence on receptor expression levels on macrophages for viral entry. As proposed to explain the higher efficiency of virus cell-to-cell transfer between CD4+ T cells across the virologic synapse, multiple mechanisms could be involved in virus cell-to-cell transfer from infected T cells to macrophages. The formation of the virologic synapse is characterized by a redistribution and clustering of HIV-1 receptors and envelope glycoproteins at the surface of the target and virus-donor cells, respectively.<sup>132,133</sup> Although similar processes in the context of infected T cell-macrophage contacts remain to be demonstrated, they could similarly reinforce envelope/receptor interactions and decrease the dependence on receptor expression levels. In addition, redistribution of coreceptors into distinct membrane environments might change their conformational states, thereby influencing envelope binding and virus entry.<sup>134</sup> Although non-macrophage-tropic envelope trimers adopt a conformation distinct from that of macrophage-tropic envelopes with a weaker affinity for CD4,<sup>135-137</sup> a local increase of viral envelope molecules at the T cell/macrophage contact site could overcome this limitation and favors cell-cell fusion. In contrast, cell-free virus particles, known to express limited amounts of Env trimers at their surface, could not overcome this limitation.<sup>138,139</sup>

Both cell-cell fusion sequential processes, first between infected T cells and myeloid cells (i.e., macrophages, iDCs, and OCs) and then with surrounding noninfected myeloid cells, are dependent on interactions of the viral envelope glycoproteins expressed on virus-donor cells with the CD4 and chemokine coreceptors expressed on myeloid
target cells. Both cell-cell fusion processes are inhibited by anti-CD4 and anti-envelope antibodies, as well as by drugs, such as T20, a specific fusion inhibitor targeting the transmembrane gp41 subunit, and also by CCR5 and CXCR4 antagonists.<sup>113,114</sup> Interestingly, no such cell-cell fusion process was observed between HIV-1-infected T cells and nondifferentiated blood monocytes or mature DCs,<sup>127</sup> even though these other myeloid cells both express CD4 and CCR5. This could be related to the lower expression of the CD4 and CCR5 receptors on the cell surface of these cell types compared with immature DCs and differentiated macrophages.<sup>127,140,141</sup> These cell-cell fusion mechanisms could be also regulated by other cell-surface molecules such as adhesins, integrins or lectins, as shown for homotypic macrophage cell fusion and OC formation.<sup>104</sup> Again, some signaling pathways involved in programming of myeloid cells into a fusion-competent state could participate in this 2-step cell-cell fusion process, for specific HIV-1 cell-to-cell spreading in these myeloid cells.<sup>82,105</sup> As mentioned above for virus cell-to-cell transfer in myeloid cells through homotypic cell-cell fusion, cellular pathways mobilized by some cytokines, such as IL-4 and IL-13, may contribute to both cell-cell fusion steps allowing virus cell-to-cell transfer and then dissemination in macrophages, iDCs and OCs. Interestingly, it was reported that some cell surface molecules, such as tetraspanin proteins (CD9, CD63, and CD81), may have a modulatory role in cell-cell fusion and formation of syncytia between infected and target CD4+ T cells.<sup>142</sup> By regulating the remodeling of actin cytoskeleton and ERM (Ezrin, Radixin, Moesin) proteins, these tetraspanins could negatively regulate cell-cell fusion between CD4+ T cells for efficient HIV-1 cell-to-cell transfer through formation of the virologic synapse.<sup>122,143-147</sup> It is not known whether such tetraspanins could differentially modulate the cytoskeleton organization depending on the target cell-type during intercellular interactions between infected CD4+ T cells and macrophages leading to cell-cell fusion for HIV-1 transfer.

Interestingly, the 2-step cell-cell fusion mechanism for massive virus cell-to-cell transfer between infected CD4+ T cells and myeloid cell targets bypasses the inhibition to HIV-1 replication related to the high expression of the SAMHD1 host cell restriction factor observed when these myeloid cells are infected with cell-free viruses.<sup>148-151</sup> In infected MGCs formed upon cell-cell fusion between infected T cells and macrophages or iDCs, SAMHD1 is expressed in its inactive phosphorylated form explaining that MGCs can survive for a long time in vitro while producing large amounts of virus particles, and this could be responsible for high productive infection of these target cells in vivo.<sup>127,152-155</sup> Although further investigations are needed to elucidate this intriguing mechanism of SAMHD1 phosphorylation in MGCs, we can speculate that the cyclin/cyclin-dependent kinase complex involved in SAMHD1 phosphorylation could be transferred from activated proliferative infected T cells through the delivery of its cellular content to the myeloid target cell. In addition, the high levels of dNTPs transferred from proliferative-infected T cells could also help to overcome the restriction activity of SAMHD1 expressed in myeloid cells. Finally, an attractive hypothesis could be related to the maintenance of transcriptionally active T cell nucleus after the initial heterotypic fusion between infected T cells and myeloid target cells (S. B. and

M. W., unpublished observations). Thus, transfer of the T cell nucleus already containing integrated proviral DNA may allow to bypass the reverse transcription step restricted by SAMHD1, leading to efficient viral expression by the newly fused cells formed. Similarly, it would be intriguing to analyze whether virus cell-to-cell transfer by cell-cell fusion could also bypass or overcome other host cell restriction factors acting through inhibition of the early steps of the HIV-1 life cycle (from virus entry until integration of the proviral genome into the host chromosomes), such as members of the APOBEC3 family, UNG2, SERINC, as well as TRIM and IFITM proteins.<sup>156-163</sup> The transfer and maintenance of the T cell nucleus containing integrated proviral DNA may also significantly decrease the sensitivity of infected MGCs to antiretroviral drugs acting in the early steps of the virus life cycle, such as inhibitors of the reverse transcriptase and integrase viral enzymes.

Again, these results may represent an original cellular mechanism for SAMHD1-independent cell-to-cell transfer of HIV-1 in myeloid cell targets through the formation of the infected MGCs observed in vivo in tissues of HIV-1-infected patients.

## 4 | CONCLUDING REMARKS

During the last decade, myeloid cells, including macrophages, DCs, and OCs, have emerged as crucial target cells of HIV-1 and likely participate in all steps of pathogenesis. Infected myeloid cells have been involved in sexual transmission and early virus dissemination in lymphoid and nonlymphoid tissues, such as brain, lung, and bones, where they can participate in the establishment of long-lived virus reservoirs even in infected patients under highly active antiretroviral therapy. Although these myeloid cells are poorly infected in vitro by cell-free viruses, the different mechanisms of virus cell-to-cell transfer recapitulated in this review (Figure 1) may represent the in vivo dominant mode of virus dissemination for productive infection of both myeloid and lymphoid cell types. As reviewed here, different mechanisms of homotypic or heterotypic virus cell-to-cell transfer between macrophages or from virus-donor-infected CD4+ T lymphocytes toward myeloid cell targets have been reported, respectively. At least in vitro, these different intercellular routes of infection of myeloid cells, and especially the mechanism related to cell-cell fusion with infected CD4+ T cells, are several orders of magnitude more efficient than infection with cell-free virus particles. These cell-to-cell infection processes may indeed bypass the inhibitory activity of host cell restriction factors, such as SAMHD1, especially in the early steps of the viral life cycle. They may also allow efficient transfer of HIV-1 strains previously defined as non-macrophage tropic, such as T/F viruses involved in sexual transmission and early dissemination in tissues. However, there is still a paucity of studies addressing the importance of HIV-1 cell-to-cell transfer in vivo even if HIV-1 cell-to-cell dissemination is believed to contribute to HIV-1 pathogenesis. Therefore, it would be of great interest to validate that the different mechanisms of HIV-1 cell-to-cell transfer described above can be recapitulated with tissue macrophages such as lung alveolar or placental macrophages,<sup>148-151,164</sup> which are particularly more resistant to infection than monocyte-derived macrophages, using ex vivo human tissue
explant models. Finally, further in vivo investigations in animal models, such as SIV-infected monkeys or HIV-1-infected humanized mice, are absolutely required for formal demonstration of the biologic relevance of this mode of intercellular virus transfer for spreading in myeloid cells in HIV-1-infected patients.

## AUTHORSHIP

Writing original draft of the manuscript: M. H., M. W., and S. B. *Editing the manuscript*: S. B., R. M., Z. V., and C. V. *Conceptualization and supervision of the review*: S. B. *Contributed equally to this paper*: M. H. and M. W. All authors have read and agreed to the present version of the manuscript.

## ACKNOWLEDGEMENTS

We want to thank Bernard Lagane and Jean-Christophe Pages for constant scientific discussions and advices. Financial support is provided by INSERM, CNRS, and the Universities of Paris and Toulouse. Both teams are also funded by grants from "Agence Nationale de Recherche sur le SIDA et les Hépatites virales" (ANRS) and "Agence Nationale de la Recherche" (ANR). M. H. is supported by the China Scholarship Council, whereas M. W., Z. V., and R. M. are supported by grants from ANRS.

## DISCLOSURES

The authors declare no conflict of interest.

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**How to cite this article:** Han M, Woottum M, Mascarau R, Vahlas Z, Verollet C, Benichou S. Mechanisms of HIV-1 cell-to-cell transfer to myeloid cells. *J Leukoc Biol.* 2022;1-11. https://doi.org/10.1002/JLB.4MR0322-737R