# LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4 + T lymphocytes

Baptiste Pradel, Guilhem Cantaloube, Marie Villares, Maika S Deffieu, Véronique Robert-Hebmann, Vincent Lucansky, Mathias Faure, Nathalie Chazal, Raphaël Gaudin, Lucile Espert

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Baptiste Pradel, Guilhem Cantaloube, Marie Villares, Maika S Deffieu, Véronique Robert-Hebmann, et al.. LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4 + T lymphocytes. Autophagy, 2024, 20 (8), pp.1825-1836. 10.1080/15548627.2024.2338573. hal-04741706

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Submitted on 17 Oct 2024

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Distributed under a Creative Commons Attribution 4.0 International License
LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4+ T
lymphocytes

Baptiste Pradel<sup>a</sup>, Guilhem Cantaloube<sup>a</sup>, Marie Villares<sup>a</sup>, Maïka S. Deffieu<sup>a</sup>, Véronique Robert-Hebmann<sup>a</sup>, Vincent Lucansky<sup>a,c</sup>, Mathias Faure<sup>b</sup>, Nathalie Chazal<sup>a</sup>, Raphaël Gaudin<sup>a</sup> and Lucile Espert<sup>a</sup>

Authors Affiliations

**Contact** Lucile Espert lucile.espert@irim.cnrs.fr Institut de Recherche en Infectiologie de Montpellier (IRIM), 1919, route de Mende, 34293 Montpellier, France

**Abstract**

HIV-1 entry into CD4+ T lymphocytes relies on the viral and cellular membranes’ fusion,
leading to viral capsid delivery in the target cell cytoplasm. Atg8/LC3B conjugation to lipids,
process named Atg8ylation mainly studied in the context of macroautophagy/autophagy, occurs
transiently in the early stages of HIV-1 replication in CD4+ T lymphocytes. Despite numerous
studies investigating the HIV-1-autophagy interplays, the Atg8ylation impact in these early
stages of infection remains unknown. Here we found that HIV-1 exposure leads to the rapid
LC3B enrichment towards the target cell plasma membrane, in close proximity with the

<footnote><sup>a</sup> University of Montpellier, 34090 Montpellier, France. CNRS, Institut de Recherche en
Infectiologie de Montpellier (IRIM), 34293 Montpellier, France</footnote>

<footnote><sup>b</sup> CIRI, University of Lyon, Inserm U1111, Claude Bernard University Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France.</footnote>

<footnote><sup>c</sup> Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin (JFMED CU), Department of Pathophysiology, Mala Hora 4C, 036 01 Martin, Slovakia.</footnote>
incoming viral particles. Furthermore, we demonstrated that Atg8ylation is a key event facilitating HIV-1 entry in target CD4⁺ T cells. Interestingly, this effect is independent of canonical autophagy as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical step of its replication cycle.

## KEY WORDS

Atg8ylation, CD4⁺ T lymphocyte, HIV-1, LC3B, virus entry

**Abbreviations:** BafA1: bafilomycin A₁; BlaM: beta-lactamase; CD4⁺ TL: CD4⁺ T lymphocytes; PtdIns3K-BECN1 complex: BECN1-containing class III phosphatidylinositol 3-kinase complex; Env: HIV-1 envelope glycoproteins; HIV-1: type 1 human immunodeficiency virus; PM: plasma membrane; PtdIns3P: phosphatidylinositol-3-phosphate; VLP: virus-like particle.

## Introduction

Human immunodeficiency virus type 1 (HIV-1) entry in CD4⁺ T Lymphocytes (CD4⁺TL) relies on different coordinated steps. Briefly, after its attachment at the surface of target cells, the viral envelope glycoprotein (Env), composed of gp120 and gp41, assembled in trimers at the surface of the viral particle, interacts with their specific receptors, CD4 and a co-receptor, CXCR4 or CCR5, depending on the viral tropism. These events lead to the fusion between the viral and cellular membranes allowing the viral capsid delivery into the cytoplasm of the target cell [1,2]. Along its journey towards the nucleus, the viral genome, which is protected by the viral capsid, is retro-transcribed into DNA by the viral reverse transcriptase. Once in the nucleus, the viral capsid is uncoated and the retro-transcribed viral genome is inserted in the target cell genome
[3-5]. After this integration step, the viral genome expression produces all the viral components necessary for the synthesis of new infectious viral particles that will mature after budding from the infected cells [6].

In a previous study, we have demonstrated that macroautophagy, evidenced by the Atg8/LC3B conjugation to lipids, is rapidly and transiently induced upon HIV-1 infection of CD4+ TL [7-9]. Macroautophagy, hereafter referred simply to as autophagy, is a major lysosomal degradation and recycling pathway involved in many cellular functions [10-12]. Notably, it is a protective mechanism against infections able to directly degrade pathogenic components or by regulating innate and adaptive immunities [13,14]. This process relies on the formation of double-membrane compartments, called phagophores, which sequester cytoplasmic components; the phagophores mature into autophagosomes that are degraded after their fusion with lysosomes. The autophagy initiation is tightly regulated by several signaling complexes such as the ULK1/ULK2-RB1CC1/FIP200-ATG13-ATG101 complex and the BECN1-containing class III phosphatidylinositol 3-kinase (PtdIns3K-BECN1) complex [13]. Autophagy is commonly monitored by following the conjugation of the Atg8/LC3B family members to a lipid, phosphatidylethanolamine (PE), because the Atg8/LC3B-PE conjugate is anchored in the phagophore and autophagosomal membranes and is degraded together with the substrates in the lysosome, making it a good marker of the autophagic flux [14]. Even if ATG (AuTophaGy related) proteins have been identified for their involvement in autophagy, it is now clear that they also exert independent functions [15,16]. In particular, the conjugation of the Atg8/LC3B family members to lipids, recently named "Atg8ylation" by analogy to ubiquitination, has been proposed to be a general mechanism involved in the maintenance of membrane homeostasis during remodeling [17,18]. Interestingly, it has been shown that the conjugation of Atg8 proteins is not restricted to PE and that this protein can also be conjugated to phosphatidylserines/PS [19,20]. Atg8ylation involves an E1-like ATG7 protein, two E2-like
ATG3 and ATG10 proteins and finally the E3-like complex ATG12-ATG5-ATG16L1. The recruitment of these ubiquitin-like conjugation systems to membranes requires the upstream production of phosphatidylinositol-3-phosphate (PtdIns3P) by the PtdIns3K-BECN1 complex [21]. It is worth noting that, so far, the ULK1/ULK2-RB1CC1/FIP200-ATG13-ATG101 complex is only required for canonical autophagy, allowing to make a clear distinction with other Atg8ylation-mediated processes [22].

During their replication, viruses have to face different cellular and immunological barriers in order to achieve a successful replication cycle either by blocking or hijacking cellular processes, in particular the autophagic machinery, to their own benefit [23]. Indeed, autophagy is rapidly activated upon viral infections and contributes to viral sensing, allowing a tight control of the infection. For example, the engagement of the measles virus (Edmonston strain) cell surface receptor CD46 induces autophagy in the early stage of infection via an indirect association with BECN1 and herpesviruses are able to stimulate autophagy in the early steps of infection before inhibiting the autophagic maturation step to avoid their degradation [24-26]. Nothing is currently known about the interplay between the autophagic machinery and HIV-1 during the early steps of CD4+ TL infection but it has been demonstrated that the downregulation of several ATGs favors HIV-1 infection in these cells [27]. This effect has also been observed through a functional genomic screen showing the pro-HIV-1 role of some ATG proteins, in particular ATG7 and ATG12, which are known to be involved in the conjugation of Atg8 proteins [28]. Consequently, the aim of our study was to determine the consequence of the early induction of Atg8/LC3B (hereafter only referred as LC3B) conjugation, observed during HIV-1 CD4+ TL, on viral replication. The results presented here show that LC3B is enriched towards the plasma membrane (PM) of the target cells, in close proximity of the HIV-1 entry sites. Moreover, we show that the conjugation machinery of LC3B favors the HIV-1 entry step in CD4+ TL independently of canonical autophagy. Altogether, our results bring
unprecedented information for the understanding of the cellular mechanism used by HIV-1 to
promote the first step of its replication cycle.

**Results**

***PtdIns3P production favors HIV-1 replication in CD4⁺ T lymphocytes.***

The Atg8-family protein LC3B is conjugated to lipids during the early steps of HIV-1
replication in CD4+ TL suggesting that HIV-1-induced autophagy could influence the viral
replication ⁷. Moreover, several ATG proteins were shown to favor HIV-1 infection [27,28].
To evaluate the role of the autophagy machinery during HIV-1 replication, we inhibited the
production of PtdIns3P, a phospholipid required for the recruitment of the Atg8ylation
machinery, by using a specific inhibitor of the PtdIns3K-BECN1 complex (PIK-III [29]). We
pre-treated CD4+ T cells, either a T cell line (HuT 78) or primary CD4+ TL purified from the
blood of healthy donors, with PIK-III during 3 h before infecting them with purified HIV-1
particles. We first verified that PIK-III did not affect cell viability or proliferation/activation of
primary CD4+ T cells in the conditions used for our experiments (data not shown). After 1 h of
contact with the viruses, target cells were extensively washed and HIV-1 replication was
monitored 16 h later by quantifying the intracellular (cell lysates) and extracellular (culture
supernatants) HIV-1 capsid protein p24 by ELISA. The results showed that the inhibition of
PtdIns3P production leads to a significant decrease in both the intracellular and the extracellular
p24 levels (Figure 1A).

After viral capsid delivery into target cells, the HIV-1 RNA genome is retro-transcribed
into DNA before being integrated in the cellular genome. To analyze the impact of PtdIns3P
production on this step of the viral replication cycle, we quantified both the early and late
reverse transcribed viral products in untreated- and PIK-III-pretreated HIV-1-infected cells. As
presented in the Figure 1B, we observed that addition of PIK-III decreased both viral reverse transcribed DNA products.

Altogether, by using two complementary approaches, we showed that PtdIns3K-BECN1 complex is required for HIV-1 replication in CD4⁺ TL, indicating that the autophagy machinery may play an important role in the early stages of the HIV-1 life cycle.

**LC3B puncta are enriched near the incoming viruses toward the target cell PM upon HIV-1-like particles exposure in a CD4-dependent manner.**

As we showed above, both p24 and viral reverse transcribed DNA products level are affected by the PIK-III pre-treatment suggesting that the autophagy machinery plays an important role in the very first steps of HIV-1 replication. In order to monitor the early viro-induced autophagic events at the single cell level, we performed live-cell imaging to analyze the dynamics of LC3B puncta formation upon virus exposure, a reliable readout to monitor the activation of the autophagy machinery. To avoid overexpression artifacts, we used a CRISPR-Cas9-based approach to generate HEK 293T cells expressing endogenous levels of a fluorescently tagged LC3B (GFP-LC3B[edit]). To this end, a sequence coding for the GFP protein was inserted upstream of the gene coding for LC3B (Figure S1A). After two rounds of cell sorting, we obtained a partially edited HEK GFP-LC3B[edit] cell line expressing endogenous levels of GFP-tagged LC3B (Figure S1B). To characterize the functionality of our model, we starved HEK GFP-LC3B[edit] cells using nutrient-free medium (EBSS), a well-known inducer of autophagy, during 4 h in the presence of bafilomycin A₁ (BafA1), which is known to impair the autophagy flux. Microscopic analyses showed that EBSS treatment expectedly resulted in an accumulation of GFP-LC3B[edit] puncta at the basal level and that this accumulation is further increased after a co-treatment with BafA1 (Figure 2A). Moreover, the LC3B puncta, revealed by the anti-LC3B antibody, colocalized with the GFP-LC3B[edit] puncta, further validating our
model for subsequent analyses (Figure 2B). This cell line was then transduced to express the
HIV-1 receptors in order to make them permissive to HIV-1 infection. Hence, we generated a
novel HEK GFP-LC3B[edit].CD4.X4.R5 cell line, which expresses CD4, CXCR4 and CCR5.
HEK GFP.LC3B[edit].X4 cells, which express only CXCR4, were also obtained for the need
of subsequent experiments (Figure S1C).

In parallel, to study the trafficking of the virus, we produced fluorescent virus-like
particles (VLPs) surrounded by an envelope expressing the HIV-1 Env at its surface (VLPs-
Env, NL4.3 strain). To do so, we produced the pcDNA3.3-Gag-mCherry construct where the
mCherry tag has been inserted between the sequence coding for the matrix protein (MA) and
the sequence coding for the capsid protein (CA). Upon maturation by the viral protease (coded
by the co-transfected PAX2 plasmid), the mCherry tag is released in the newly synthesized viral
particle but is not associated with the mature capsid/viral core. This construct allowed us to
track the viral particles and, at the same time, to monitor the viral entry step because, upon viral
and cellular membranes' fusion, the mCherry tag is released, and becomes diffused, in the
cytoplasm of the target cell. These VLPs-Env were used to mimic an HIV-1 infection of the
HEK GFP-LC3B[edit].CD4.X4.R5 cells and follow the interplay between viral particles and
GFP-LC3[edit] puncta dynamics by live-cell imaging. As shown in Figure 2C, Figure 2D and
in Movie S1, this strategy allowed us to observe an increase in the number of GFP-LC3B[edit]
puncta in VLPs-Env-exposed cells overtime. Strikingly, we noted an enrichment of these GFP-
LC3B[edit] puncta towards the target cell PM, as it is already observable in the Figure 2C after
30 min of VLPs-Env exposure. To quantify this enrichment, we stained the target cells with the
Sir-Actin dye. This dye, which stains the subcortical polymerized actin, allowed us to visualize
the cell border and, then, to measure the distance between each GFP-LC3B[edit] puncta inside
the target cell from the PM. The data presented in Figure 2E showed a significant decrease in
this distance upon VLPs-Env exposure, confirming the viral-induced enrichment of GFP-
LC3B[edit] puncta near this zone of the target cells. To control that the quantified decreased
distance between the VLPs-Env-induced GFP-LC3B[edit] puncta and the cell border was not
the result of their overall increase in the cell, we did the same experiment in cells treated or not
with trehalose, which is a well-known inducer of autophagy. As expected, trehalose treatment
induced a significant increase in the number of GFP-LC3B[edit] puncta but did not induce a
difference in their mean distance to Sir-Actin (Figure S2A) arguing for a specific-event linked
to VLPs exposure. Upon interaction with its target cells, the gp120 subunit of Env interacts first
with CD4, leading to a conformational change allowing its subsequent interaction with the co-
receptor. This last interaction allows the gp41 subunit of Env to insert in the target cell PM to
achieve the fusion between the viral and the target cell membranes, ultimately leading to the
viral capsid entry [2,30]. In order to determine which of these events is involved in the viro-
induced enrichment of LC3B puncta towards the target cell PM, we first used AMD3100, an
antagonist of CXCR4 that blocks its interaction with gp120. We verified that this compound
alone did not affect the mean distance between LC3B puncta from the Sir-Actin staining (data
not shown). Analyses of the distance between GFP-LC3B[edit] puncta and the Sir-Actin
staining revealed that the addition of AMD3100 did not prevent the viro-induced enrichment
of GFP-LC3B[edit] puncta towards the target cell PM (Figure 2F). To test the involvement of
CD4, the same experiment was done in edited target cells that do not express this receptor (HEK
GFP-LC3B[edit].X4, Figure S1C). In these conditions, the viro-induced enrichment of LC3B
puncta towards the PM was abolished (Figure 2G). This result is likely due to the lack of CD4
rather than CCR5 since the VLPs-Env do not use CCR5 for entry. Consistently, the same result
was obtained in target cells that do not express CD4 and are treated with AMD3100 (Figure
S2B). Altogether, these results strongly suggest that the gp120-CD4 interaction is involved in
the enrichment of LC3B puncta towards the target cell PM.
To go deeper in the characterization of the VLPs-Env-induced enrichment of LC3B puncta towards the target cell PM and to determine if this enrichment specifically occurs near the viral particle entry sites, we tracked the fluorescent-tagged VLPs-Env together with GFP-LC3B[edit] puncta by live-cell imaging. To achieve this goal, we delimited a zone, by drawing a sphere of 1.5 µm of diameter at the surface of the target cell before the arrival of the VLPs-Env. We analyzed the mean number of GFP-LC3B[edit] puncta in this zone, before, during and after the presence of the VLPs-Env at the cell surface, by tracking VLPs-Env particles over time. The disappearance of the mCherry signal means that the virus has entered into the target cell. As shown in Figure 2H and in the associated Movie S2, no GFP-LC3B[edit] puncta were observed at the cell surface in absence of the VLPs-Env in the delimited zone. Then, a concomitance between PM-proximal GFP-LC3B[edit] puncta and the VLPs-Env fluorescence intensity appeared in the delimited zone during the presence of the viral particles at the target cell surface. It has to be noted that, even if they appeared significantly only in the presence of the viral particles in the delimited zone, the GFP-LC3B[edit] puncta are not stably present in this zone all along the timeframe. Finally, both the VLPs-Env fluorescence intensity and the GFP-LC3B[edit] puncta disappeared after the viral particle entry. This result was quantified by tracking several fluorescent viral particles. It is important to note that the GFP-LC3B[edit] puncta enrichment is occurring near the virus and not necessarily colocalize with the virus before entry, suggesting that a direct interaction between LC3B and the incoming viral particle is not required to observe this enrichment near the viral entry site. The results were reproduced after infecting the edited cells with X4 or R5-tropic fluorescent wild-type HIV-1 (Figure S2C and S2D and associated Movie S3 and S4, respectively). Altogether, these results indicated that LC3B puncta are preferentially enriched towards the virus entry sites independently of the co-receptor used.
**LC3B conjugation favors HIV-1 entry in CD4⁺ T cells independently of the canonical autophagy pathway.**

Our results suggested that LC3B could play an important role in the HIV-1 entry step. To test this hypothesis, we first analyzed the impact of PtdIns3P production on this step of the viral replication cycle. To this aim, we used a standard method, which is the beta-lactamase (BlaM)-Vpr assay [31]. It consists in producing virions having incorporated the BlaM fused to the HIV-1 Vpr protein, which is enclosed in the viral particles. These virions were purified and used to infect CD4⁺ T cells stained with the fluorescent CCF2, which is the substrate of BlaM. Consequently, upon HIV-1 entry, the BlaM-Vpr fusion protein, released in the target cell cytosol, cleaved the CCF2 substrate. As CCF2 emits a different fluorescence upon cleavage, it allows the quantification of HIV-1 entry, occurring after the fusion between viral and cellular membranes, by flow cytometry. As shown in the Figure 3A, PIK-III pretreatment led to a significant decrease in HIV-1 entry in primary CD4⁺ TL, demonstrating that PtdIns3P production is involved in HIV-1 entry in target CD4⁺ T cells. Importantly, we validated that the drug did not affect the expression level of the HIV-1 receptor and co-receptor, CD4 and CXCR4, at the cell surface (Figure S3A). As PtdIns3P is required for the recruitment of the LC3B conjugation machinery and because we previously observed a rapid LC3B lipidation upon HIV-1 infection of CD4⁺ T cells ⁷, we next investigated the role of this process during the HIV-1 entry step. To achieve this goal, we transduced primary CD4⁺ TL with shRNAs allowing the downregulation of the expression of BECN1, which is involved in PtdIns3P production, ATG7 or ATG3, which are essential for the conjugation of LC3B to lipids. Then, shRNAs-transduced cells were infected with BlaM-VprR-containing HIV-1 and the level of viral entry was analyzed by flow cytometry as above. The results obtained showed that both the downregulation of BECN1, ATG7 and ATG3 expression leads to a decreased HIV-1 entry in target cells (Figure 3B) arguing for a role of LC3B conjugation in the observed effect. In order
to confirm the critical role of LC3B lipidation in HIV-1 entry into target cells, we did the same
BlaM-Vpr assays in CD4+ T cells overexpressing LC3B or a LC3B-mutated form unable to be
lipidated (LC3B<sup>G120A</sup>). As shown in Figure 3C, LC3B overexpression increased HIV-1 entry in
target cells whereas the mutated LC3B, incapable of lipidation, had no effect on this first step
of the viral life. These results suggest that HIV-1 particle fusion with target cell membranes is
dependent on the intracellular lipidation function of LC3B.

Nowadays, Atg8ylation is well-known to be involved in several processes besides the
canonical autophagy pathway and it is proposed to be a general membrane stress and
remodeling response [32]. In order to determine if the effect of Atg8ylation on HIV-1 entry is
due to canonical autophagy or an independent process, we analyzed this step using a cocktail
of lysosomal inhibitors targeting CTSB (cathepsin B) and CTSD (E64d + pepstatin A). As
shown in Figure 3D, inhibition of lysosomal degradation led to an increase in HIV-1 entry in
CD4+ T cells. These data are in accordance with previous reports showing that inhibition of
endosome acidification with BafA1, a drug also known to inhibit autophagic degradation,
enhances entry by fusion at the PM [33]. To specifically address the role of canonical autophagy
during HIV-1 entry, we transduced shRNAs to downregulate ATG13 expression in primary
CD4+ T cells and we tested the effect of this downregulation on HIV-1 entry using the BlaM-
Vpr assay. Indeed, ATG13, which is part of the ULK1/ULK2-RB1CC1/FIP200-ATG13-
ATG101 signaling complex, is known to be only involved in the initiation of canonical
autophagy allowing to discriminate between the role of LC3B conjugation in canonical
autophagy or in an independent process. Interestingly, we observed that the downregulation of
ATG13 led to an increase of HIV-1 fusion to target cells (Figure 3E), an opposite effect than
the one observed with shRNA targeting *BECN1*, *ATG7* or *ATG3*. Taken together, these results
show that the conjugation of LC3B to lipids participates in HIV-1 entry in target cells
independently of an autophagy-mediated degradation.
Discussion

HIV-1 entry in its target cells is the first step of a successful replication cycle. Our results show that, even if the mechanisms driving HIV-1 entry have been extensively studied since the discovery of this virus forty years ago, new technical approaches can bring additional insights in this highly dynamic process. We demonstrated, for the first time, that Atg8ylation can influence the entry step of a virus in its target cells. By using live-cell imaging, we showed that the LC3B protein is enriched towards the target cell PM upon HIV-1 exposure, particularly near the virus entry sites. In particular, the conjugation of this protein to lipids, a process recently named Atg8ylation, favors HIV-1 entry independently of the canonical autophagy process. Indeed, we observed that the downregulation of ATG13 led to no decrease, but instead enhancement, of HIV-1 fusion with target cells. One hypothesis that could explain this phenotype is that releasing the autophagic machinery components from their role in canonical autophagy would allow the Atg8ylation actors to be more available to act on viral entry. Our results are in accordance with a previous study showing that peptides derived from the ATG protein BECN1 and BECN2 (Tat-BECN1 and Tat-BECN2, respectively) favor lentivirus entry into target cells. These peptides, which have been first developed as canonical autophagy inducers [34], exert their pro-entry effect at doses that do not induce canonical autophagy but leads to LC3B conjugation [35].

Interestingly, our results showed an increase in HIV-1 entry after the addition of lysosomal cathepsins inhibitors (Figure 3D), indicating that the lysosomal degradation could restrict HIV-1 entry. However, it is well known that using these inhibitors leads to LC3B-positive membranes accumulation <sup>14</sup>. Thus, lysosomal inhibitors treatment could increase the quantity of LC3B-conjugated membranes that would be available for HIV-1 fusion process. However, we cannot exclude that several lipidation sites could be involved. Indeed, our data do
not allow us to determine if the LC3B lipidation occurs locally, directly on lipids in the vicinity
of the PM, or on lipids from intracellular membranes that would subsequently be redirected
towards viral entry sites. In addition, it would be interesting to determine if some proteins from
the endoplasmic reticulum are localized at the PM during HIV-1 entry because it has been
shown that LC3B lipidation could be driven by a local synthesis of PtdIns3P at the level of ER-
PM contact sites [36,37]. Concerning the PM lipid composition needed for HIV-1 entry, despite
numerous discrepancies, several studies pointed out the roles of cholesterol and ordered lipid
domains in HIV-1 cellular entry and indicate that viral fusion occurs preferentially through
cholesterol-rich heterogeneous membranes and not through uniformly fluid membranes
[38,39]. Thereafter, it has been shown that ordered and disordered lipid domains coexistence is
required for a successful fusion step [40]. Thus, it would be interesting to analyze if LC3B
lipidation could influence locally the lipids composition and organization at the PM and modify
its fluidity and stiffness.

At the molecular level, the interaction of HIV-1 Env with CD4 appears to be required
for the LC3B enrichment at the target cell PM suggesting a role of signaling events downstream
of this receptor. In this context, it has been reported that the RAC1 signaling complex, involved
in actin cytoskeleton remodeling and known to be activated by HIV-1 Env, promotes HIV-1
entry into target cells by acting on membrane fusion [41]. In parallel, another study showed that
infection by *Campylobacter jejuni* leads to a RAC1-dependent recruitment of LC3B at its host
cell entry site, and that the conjugation of this protein favors bacterial invasion [42]. These
results suggest that the LC3B enrichment towards the PM could be a more general pathogen-
induced phenomenon, independent of a specific cellular receptor. Consequently, other cellular
partners shall be sought for.

Atg8 is unique in yeast but seven homologs have been identified in mammals. These
proteins are classically divided in two families, which are the LC3 family, composed of LC3A,
LC3B, LC3B2 and LC3C, and the GABARAP (GABA type A receptor-associated protein) family composed of GABARAP, GABARAPL1 and GABARAPL2. Several reports demonstrated a specific role of Atg8 proteins in mediating membrane fusion. Indeed, *in vitro* studies demonstrated that the fusion between two liposomes is favored by the expression of the yeast Atg8 at their surface. The authors also observed an enrichment of these Atg8s at the junction between the liposomes [43]. Accordingly, another study, using the mammals Atg8s LC3B and GABARAPL2, showed that liposomes-bound Atg8s can induce their fusion [44]. Recently, it has been demonstrated that both the yeast Atg8 and LC3B can trigger membrane perturbations suggesting a role of these proteins in membrane remodeling [45]. Considering the amount of data in the literature regarding the contribution of the Atg8 proteins on membrane fusion, it would be important to decipher their specific actions, individually or in cooperation with each other, on fusion mechanisms taking place at the PM during HIV-1 entry.

To our knowledge, despite several examples described of Atg8ylation in cellular secretion, such as unconventional secretion or viral release [46-51], the present study is the first to identify such a role during internalization of an exogenous cargo. This discovery could have wide repercussions on the characterization of different cell biology processes taking place at the PM, such as, for example, extracellular vesicles entry in cells. In the case of HIV-1 infection, it would be critical to identify the cellular and viral partners of LC3B in the context of viral entry as the discovery of new actors in this process could lead to the development of new combined therapies in the future.

# Materials and Methods

## Cells

Primary CD4+ TL were isolated from peripheral blood mononuclear cells and purified by negative selection using the CD4+ T cell isolation kit (Miltenyi Biotec, 130-096-533). Cells
were phenotypically analyzed and activated using anti-CD3-anti-CD28 coated plates (BioXCell, BE0001-2 and BE0248, respectively,) followed by IL2 treatment (100U:mL; Miltenyi Biotec, 130-097-746). All experiments involving primary cells were performed at least 4 days post-activation. Primary CD4+ TL and HuT 78 cells were cultured and maintained in RPMI 1640 medium (Gibco, 61870-010) supplemented in 10% fetal calf serum, 1% penicillin-streptomycin. HEK 293T cells were cultured and maintained in Dulbecco's modified Eagle's medium (Gibco, 61965-026) supplemented with antibiotics and 10% fetal calf serum.

Obtention of edited cells (HEK GFP.LC3B[edit].X4 and HEK GFP-LC3B[edit].CD4.X4.R5): The HEK.GFP.LC3B[edit].X4 was generated according the method in [52]. Briefly, we transfected HEK 293T cells with a combination of a donor plasmid (p-GFP: MAP1LC3B-mEGFP gifted from Allen Institute for Cell Science; Addgene, 101783), a purified PCR product coding for the gRNA-tracrLC3B and a plasmid coding for *Streptococcus pyogenes* Cas9 (pCas9) using JetPrime (Polyplus Transfection, 101000046). Seven days post-transfection, cells were sorted to be enriched using the cell sorter Aria IIU (Becton Dickinson). A second cell sorting was performed to obtain several clones selected according to the GFP expression levels and a single clone (HEK.GFP.LC3B[edit].X4) was selected. A genomic DNA extraction was performed on this clone using the Quick DNA MicroPrep kit (Zymo Research, D3020) using the manufacturer's instructions and a subsequent PCR analysis was done using primers amplifying both the *LC3B* gene and the GFP-edited copy of this gene using Taq Platinum Polymerase Kit (Invitrogen, 10966034) (primers sequence: 5'-GCGGGCTGAGGAGATACAAG-3' and 5'-TCCGAGAGGTCCGCAGG-3'). PCR products were run on a 1% agarose gel and imaged with SYBR Safe DNA gel stain (Invitrogen, S33102) using the Axygen Gel Documentation System. The edited cells were then transduced with
lentivectors expressing CD4 and CCR5 in order to obtain the HIV-1 permissive cells named
HEK GFP-LC3B[edit].CD4.X4.R5.

Reagents and antibodies

PIK-III was purchased from Euromedex (SE-S7683); AMD3100 (A5602), BafA1 (B1793), trehalose (T0167), pepstatin A (77170) and E64d (E8640) were purchased from Sigma-Aldrich. Sir-Actin was purchased from Tebu-Bio (251SC001). The anti-LC3B (L7543), the anti-ATG7 (10088-2-AP), the anti-BECN1 Ab (11306-1-AP) and the anti-ATG3 Ab (11262-2-AP) were purchased from Proteintech. The anti-ATG13 (13468) was purchased from Cell Signaling Technology. The anti-TUBA/α-tubulin was purchased from Sigma-Aldrich (T5168). The anti-CD4-FITC antibody (130-114-722) and the anti-CXCR4-PE (130-117-504) were purchased from Miltenyi Biotec and the anti-CCR5 (2D7) from Becton Dickinson.

Viruses

X4-tropic viruses: HEK 293T cells were transfected with pNL4-3 (obtained from the NIH HIV Reagent Program ARP-114) using PEImax (Clinisciences, 24765). X4-tropic BlaM-Vpr viruses: HEK 293T cells were co-transfected with pNL4.3 and pBlaM-Vpr (kindly provided by Caroline Goujon, IRIM UMR 9004 CNRS UM, Montpellier). Viral particles were concentrated by ultracentrifugation on a PBS (Gibco, 14190-094)-25% sucrose (Millipore, 84100) cushion after the collection of the culture supernatants 48 h post-transfection. post-transfection. The infectivity of those viruses was titrated using JLTRG (obtained from the NIH HIV Reagent Program, ARP11586). Fluorescent X4-tropic viruses: HEK 293T cells were co-transfected with pNL4.3 and pcDNA3.3-Gag-mCherry (Addgene, 208882; deposited by Raphaël Gaudin). This last plasmid was obtained as followed: Fragment Gag-mCherry modified with restriction sites NheI at 5'-end and PmeI at 3'-end was amplified using the following primers NheI-Gag-F (5' gactGCTAGCgccaccatgggtgcgagagcgtcggtattaag 3') and Gag-PmeI-R (5' ccccGTTTAAACttattgtgacgaggggtcgctgc 3'). The template for the PCR reaction was pNL4.3
bearing the Gag sequence labeled with mCherry. Both produced fragment and vector
pcDNA3.3 were treated with restriction enzymes. *Fluorescent R5-tropic viruses:* HEK 293T
cells transfected with the pNL4.3.Gag-imCherry molecular clone (kindly provided by Philippe
Benaroch, Institut Curie INSERM U932, Paris), which carries an R5-tropic envelope thanks to
a mutated gp120 V3-loop [53]. *VLPs-Env:* HEK 293T cells were co-transfected with
pCDNA3 Env (from NL4.3), psPAX2 (Addgene, 12260a; deposited by Didier Trono),
pcDNA3.3-Gag-mCherry and pLenti-GFP. Quantification of viral particle production was
performed with a p24 enzyme-linked immunosorbent assay (ELISA) from Innogenetics
(Innotest HIV antigen monoclonal Ab [MAb]; 80563) according to the manufacturer's
instructions.

Lentiviruses production and transduction

HEK 293T cells were co-transfected with pCMV-VSV-G, psPAX2 and pLKO.1-puro shRNA-expressing plasmids (Sigma-Aldrich, MISSION shRNA, shATG13: TRCN0000172801, shATG3: TRCN0000146846, shATG7: TRCN0000007584, shBECN1: TRCN0000299864, shCT: 012120009NM). Produced lentiviruses were concentrated and quantified as previously.

To obtain the GFP constructs-expressing lentiviruses, HEK 293T cells were co-
transfected with pCMV-VSV-G (Addgene, 12259; deposited by Didier Trono), pPAX2 and
pNaldini-eGFP [54] or pNaldini-eGFP-LC3B (provided by Mathias Faure). The pNaldini-
eGFP-LC3B<sup>G120A</sup> was obtained by directed mutagenesis using the Q5 site directed mutagenesis
kit (New England Biolabs, E0554S). Produced lentiviruses were concentrated and quantified as
previously. Target CD4<sup>+</sup> T cells were transduced with plasmids encoding GFP, GFP-LC3B or
GFP-LC3B<sup>G120A</sup> lentiviral particles (ratio of 4:1 between GFP and GFP-LC3B or GFP-
LC3B<sup>G120A</sup> constructs to obtain the same percentage of transduction) during 72 h before
experiments.

Analysis of *HIV-1* reverse transcribed products by qPCR
HuT 78 cells or primary CD4⁺ T cells were infected with DNase I-pretreated WT-X4-tropic viruses for 16 h. DNA from infected cells was extracted using the QIAamp DNA-minikit (Qiagen, 51304) according to the manufacturer's instructions. Quantitative PCR of early and late retro-transcribed products was done using the TaqMan Universal Master mix II no UNG (ThermoFisher, 4440040,) and the quantity normalized to PGBD loading control. Analysis was performed with LightCycler 480 with the method of 2ΔΔCT.

### Western blot

Cell pellets were lysed in 2x Laemli Buffer and heated 10 min at 95°C. Samples were loaded on 12% Prosieve 50 gels (Lonza, LON50618) and transferred to polyvinylidene/PVDF membranes. After a blocking step in Tris-buffered saline (TBS composed of 50 mM Tris-HCl, pH 7.5 and 150 mM NaCl) containing 5% casein (Sigma-Aldrich, C7078) and 0.05% Tween 20 (Sigma-Aldrich, P9416) for 1 h at room temperature, blots were incubated overnight at 4°C with the primary Ab in the blocking buffer. After 3 washes with TBS + 0.05% Tween 20, the blots were incubated for 1 h at room temperature with peroxidase-coupled antiserum (Sigma-Aldrich, goat anti-rabbit and goat anti-mouse, A0545 and A2304, respectively) diluted in blocking buffer. After further washes, the immune complexes were revealed by ECL (Clarity western ECL Substrate; Bio-Rad, 170-5061) and analyzed with a ChemiDoc camera (Bio-Rad). Quantification of protein expression was performed using the Image lab Software (Bio-Rad). Data were normalized with reference to the densitometry analysis of TUBA.

### BlaM-Vpr assay

Target cells were infected with X4-tropic BlaM-Vpr viruses at a MOI of 1 for 3 h. After a wash with a CO2-independent media (ThermoFisher, 18045088), cells were loaded with CCF2 (ThermoFisher, K1032) for 1 h 30 min at room temperature (RT). Cells were then washed with CO2-independent media and the BlaM reaction was developed overnight at RT. Finally, the infected cells were washed once in PBS, fixed with formalin (Sigma-Aldrich, HT501128) and
analyzed using flow cytometry using the NovoExpress software. For experiments with GFP-LC3B and GFP-LC3B<sup>G120A</sup> transduced cells, a compensation was required and varied depending on levels of transduction. Thus, the entry levels were monitored using the value of cleaved-CCF2 rStaining using the following formula: rStain = (MFI CCF2-cleaved in infected cells - MFI CCF2-cleaved in uninfected cells):(rSD MFI CCF2-cleaved in uninfected cells).

## Live-cell imaging

One day prior imaging, edited cells were seeded in an Ibidi 35-mm high plate (Clinisciences, 81156) and grew overnight at 37°C, 5% CO₂. Then, the cells were pretreated during 90 min with an anti-bleaching media (Gibco, DMEM without phenol red; 31053023) containing 1% penicillin-streptomycin, 10% FBS and 1% Prolong Live Antifade Reagent (ThermoFisher, P36975). Sir-Actin staining was performed during 15 min at 0.5 µM and removed before infection. Cells were infected with viral particles for 30 min at 16°C for synchronization, then put 10 min in the slide chamber at 37°C to allow the following proper imaging. Movies were acquired using an inverted confocal microscope Nikon coupled with Spinning Disk Dragonfly Andor at an 60x objective. 488 nm, 561 nm and 637 nm channels were acquired (GFP-LC3B, GagmCherry, Sir-Actin respectively) every 10s for 30 min by focusing on the top of the cells. This technology allowed us to capture several z-stack (13 z-stacks each separated of 0.5 µm).

## Quantification of the distance between LC3B and the plasma membrane

Individual cells, selected using the Sir-Actin staining, were cropped using the Fiji software. Each cell was imported in the IMARIS software to quantify the distance between GFP-LC3B[edit] puncta and plasma membrane in 3 dimensions. Briefly, we identified the plasma membrane (Surface module) using the SirActin channel and applied a manual threshold with background subtraction and obtained a surface highlighting the cell contouring. Then, using the "Spots" module, we detected the GFP-LC3B puncta and we exported the measures "Shortest
distance to Surface", which indicate the distance of each GFP-LC3B[edit] puncta from the
plasma membrane.

Quantification of the GFP-LC3B[edit] puncta toward the viruses at the cell surface

The upper stacks (1.5 µm of depth acquired within 3 z-stacks separated by 0.5µm) were studied by generating a z-projection (based on maximum intensity). We captured viral particles by choosing blindly, using the GagmCherry and SirActin stainings, the incoming Gag.mCherry puncta at the plasma membrane that, lately, entered into the target cell. Tracking was performed manually by delimiting an arbitrary zone of 1.5 µm diameter around the viral particle, independently of its size, at each timeframe. Then, for every timepoint, we counted the number of GFP-LC3B puncta in the delimited zone using the “Analyze particles” module after having applied a manual threshold determined for each cell and maintained throughout the timeframes.

Therefore, for every viral particle tracked, we obtained a mean value of the GFP-LC3B puncta number in the following 3 conditions: “Before virus”, during “Virus” tracking and “After virus”.

Statistical Analysis

Statistical analyses were performed using Graph Pad Prism software version 8. Differences
were considered significant at *p*<0.05, **p**<0.01, ***p*<0.001. Specific statistical treatments
are described in the figure legends.

**Declaration of interests**

All authors declare they have no competing interests.

Acknowledgments

We thank Dr. Philippe Benaroch (Institut Curie INSERM U932, Paris) for the Env-mutated
pNL4.3.Gag-mCherry (R5 tropic). We thank Dr. Caroline Goujon (IRIM UMR 9004 CNRS
UM, Montpellier) for having provided us the pBlaM-Vpr plasmid. The following reagent was obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: Jurkat LTR-GFP CCR5⁺ Cells (JLTRG-R5), ARP-11586, contributed by Dr. Olaf Kutsch. We acknowledge the imaging facility MRI, member of the national infrastructure “France-BioImaging infrastructure” supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»)”. We are grateful to S. Lyonnais and the CEMIPAI BSL-3 facility for the HIV-1 live-cell imaging experiments. We warmly thank Sam Howells for the manuscript proofreading. This work was supported by the “ANRS - Maladies Infectieuses Emergentes », by SIDACTION, by the Centre National de la Recherche Scientifique (CNRS) and by the University of Montpellier.

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## Figures

**Figure 1.** PtdIns3K inhibition decreases HIV-1 infection in CD4⁺ T lymphocytes. (A) HuT 78 cells or primary CD4⁺ TL were pre-treated with 20 µM PIK-III for 3 h before addition of HIV-1 (NL4.3 strain) at M.O.I. 1 during 1 h at 4°C. HIV-1-exposed cells were then extensively washed to remove the unbound viruses and the drug before being cultured for 16 h at 37°C. p24 levels were quantified by ELISA on cell lysates and supernatants. The p24 levels in cell lysates was normalized to the total level of protein extract. Statistical analyses were performed using the unpaired t test with Graph Pad Prism software version 8. (B) HuT 78 cells or primary CD4⁺ TL were pre-treated with 20 µM PIK-III for 3 h before extensive washings to remove the drug. Cells were then infected with Dnase I-treated HIV-1 (NL4.3 strain) at M.O.I. of 1 for 16 h at 37°C. Early and late RT DNA intermediates were quantified by qPCR and normalized on PBGD levels. Analysis was performed using the 2-ΔΔCt⁺. Statistical analyses were performed using the unpaired t test with Graph Pad Prism software version 8.

**Figure 2.** LC3B puncta are enriched toward the target cell PM, near the incoming viruses, upon HIV-1-like particles exposure in a CD4-dependent manner. (A) HEK GFP-LC3B[edit].CD4.X4.R5 cells were treated with EBSS in combination, or not, with 50 nM BafA1 for 4 h. The total volume of the cells was acquired using a confocal microscope and the number of GFP-LC3B[edit] dots were analyzed using the Plug in « Analyze particles » of the Fiji software in each condition. The data are representative of 3 independent experiments. Statistical analyses were performed using the unpaired t test with Graph Pad Prism software version 8. (B) EBSS-treated edited cells, in combination with BafA1 treatment, were stained with an anti-LC3B antibody for immunofluorescence. The percentage of colocalization between GFP-LC3B[edit] puncta and LC3B puncta detected by immunofluorescence was analyzed (Manders' coefficient of the green fluorescence in the red fluorescence). At least 30
cells from 3 independent experiments were analysed. The statistical analysis was done using
the "one sample t and wilconxon test" on Prism 8 software. (C) HEK GFP-
LC3B[edit].CD4.X4.R5 cells were incubated with mCherry-VLPs-Env and live-imaged using
a Spinning Disk Dragonfly microscope. A snapshot after 30 min of mCherry-VLPs-Env (Red)
exposure is presented. The white arrows point to the GFP-LC3B[edit] (green) puncta that
appeared towards the cell surface. (D) HEK GFP-LC3B[edit].CD4.X4.R5 cells were incubated,
or not, with mCherry-VLPs-Env during 90 min and acquired using a confocal microscope. The
number of GFP-LC3B[edit] puncta were analyzed in the total volume of the cells using the
« Analyze particles » plugin of the Fiji software in each conditions. The data are representative
of 3 independent experiments. Statistical analyses were performed using the unpaired t test with
Graph Pad Prism software version 8. (E) HEK GFP-LC3B[edit].CD4.X4.R5 cells were stained
with 0.5 µM SirActin and, then, infected with mCherry-VLPs-Env. After 20 min of virus
exposure, the total volume of the cells was imaged with a spinning disk confocal microscope.
Red: SirActin staining; Green: GFP-LC3B[edit]. The graph represents the average distance
between GFP-LC3B[edit] puncta and the SirActin staining per cell. Analysis was performed
with the IMARIS software, using the "Surface" function to identify the cell border and using
the "Spots" function to identify individual GFP-LC3B[edit] puncta. The average distance
between GFP-LC3B[edit] puncta and the SirActin staining was quantified in at least 20 cells
from at least 3 independent experiments. One single representative z is presented for each
condition. Statistical analyses were performed using the unpaired t test with Graph Pad Prism
software version 8. (F) The same experiment as in (C) was done in HEK GFP-
LC3B[edit].CD4.X4.R5 cells pretreated during 90 min with 1 µg:ml AMD3100. Data are
representative of at least 20 cells from 3 independent experiments. (G) The same experiment
as in C was done using the HEK GFP-LC3B[edit].X4 cells that do not express CD4. Data are
representative of at least 20 cells from 3 independent experiments. (H) HEK GFP-
LC3B[edit].CD4.X4.R5 were exposed to mCherry-VLPs-Env during 30 min at 16°C. Then, viruses-exposed cells were incubated at 37°C and three-dimensional images were acquired every 10 s during 30 min using a spinning disk confocal microscope. Images were captured with a focus on the top of the cell. Viruses were tracked at the surface of the target cells based on the SirActin staining. A sphere of 1.5 µm of diameter was drawn around each virus (yellow circle) and the number of LC3B puncta found in this volume was evaluated using the “FiJi” software at each time point. The quantification was done in three conditions: (i) at the surface of the target cell in the zone where the virus will arrive (before virus), (ii) in the sphere volume along the presence of the virus at the PM (virus) and (iii) in the sphere after the virus entry (after virus). Snapshots of a representative individual tracked viral particle over time is shown for each condition. The left graphs represent the correlation between the Gag.mCherry intensity in the sphere (red) and the number of LC3B puncta (green) in the same volume. The right bar graphs summarize the mean value of the LC3B puncta number in the sphere for at least 10 viruses in at least 3 independent experiments. Data are representative of at least 20 cells from 3 independent experiments.

**Figure 3.** LC3B conjugation machinery favors HIV-1 entry by fusion in CD4⁺ T lymphocytes in an autophagy-independent manner. (A) Activated primary CD4⁺ TL were pre-treated with either 0.1% DMSO or 20 µM PIK-III for 3 h then washed intensively to remove drugs. Pre-treated cells were infected with HIV-1.Blam-Vpr (M.O.I. 1) for 3 h at 37°C. After washing, cells were loaded with CCF2 for 1.5 h at RT before being washed and incubated overnight at RT to allow Blam enzymatic reaction. Flow cytometry was used to quantify the percentage of cells displaying an increased blue fluorescence representative of viral entry. Data are representative of at least 3 independent experiments. Statistical analyses were performed using the unpaired t test with Graph Pad Prism software version 8. (B) Activated primary CD4⁺ TL
were transduced with shRNA targeting *BECN1* or *ATG7* for at least 3 days. Transduced cells
were directly lysed in 2x Laemmli loading buffer to analyze the expression level of *BECN1*
and *ATG7* by western blot. α-Tubulin (TUBA) was used to control sample loading. Transduced
cells were infected with HIV-1-Blam-Vpr as previously and the viral entry was analyzed as in
A. Data are representative of at least 3 independent experiments. Statistical analyses were
performed using the unpaired t test with Graph Pad Prism software version 8. (C) HuT 78 cells
were transduced for 3 days with lentiviruses allowing the expression of either GFP, GFP-LC3B
or GFP-LC3B<sup>G120A</sup> before being infected with HIV-1-Blam Vpr using the same protocol of
infection as A and B. For this experiment, as compensation was required and varied depending
on the transduction levels. The entry level was monitored using the value of relative staining
(rStain) of cleaved-CCF2 in infected conditions using the following formula: rStain = (MFI
CCF2-cleaved infected-MFI CCF2-cleaved uninfected):(rSD MFI CCF2-cleaved uninfected).

Data are representative of at least 3 independent experiments. Statistical analyses were
performed using the unpaired t test with Graph Pad Prism software version 8. (D) Activated
primary CD4+ TL were pre-treated with either 0.1% DMSO or Antiproteases (AP = Pepstatin
A + E64d, 10µM each) for 3 h, then washed to remove drugs. Then cells were infected with
HIV-1 NL4.3 Blam-Vpr using the same protocol of infection and analysis as in A. Data are
representative of at least 3 independent experiments. Statistical analyses were performed using
the unpaired t test with Graph Pad Prism software version 8. (E) Activated primary CD4+ TL
were transduced with shRNA targeting *ATG13* for 3 days before being directly lysed in 2x
Laemmli loading buffer to analyze the expression level of *ATG13* by western blot. TUBA was
used to control sample loading. Transduced cells were infected with HIV-1-Blam-Vpr using
the same protocol of infection and analysis as in A. Data are representative of at least 3
independent experiments. Statistical analyses were performed using the unpaired t test with
Graph Pad Prism software version 8.