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Productive HIV infection in astrocytes can be established via a nonclassical mechanism

Li, Guan-Hana; Maric, Draganb; Major, Eugene O.c; Nath, Avindraa

Author Information

aSection of Infections of the Nervous System

bFlow and Imaging Cytometry Core Facility

cLaboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.

Correspondence to Dr. Guan-han Li, Bldg 10, Room 7C-103, National Institutes of Health, Bethesda, MD 20892, USA. Tel: +1 301 402 0840; e-mail: [email protected]

Received 27 October, 2019

Revised 21 January, 2020

Accepted 3 February, 2020

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).

doi: 10.1097/QAD.0000000000002512

Abstract

Introduction

In recent years, a major effort has been focused on developing strategies to eradicate HIV from tissue reservoirs or achieving a functional cure by maintaining the virus in a nonreplicating or latent state [1–6]. However, major setbacks to these approaches have occurred because of re-emergence of HIV from the reservoirs. The brain has been proposed as an important HIV reservoir [7–10]. However, the mechanisms by which this reservoir is established and maintained are poorly understood.

Although the incidence of HIV-associated dementia has decreased considerably with the use of combined antiretroviral therapy (cART), the prevalence of milder forms of HIV-associated neurocognitive impairment is rising [11–15]. This is partially attributable to increased survival, but a persistent brain reservoir may also play a critical role. Perivascular macrophages and microglia are major cell types infected with HIV in the central nervous system (CNS) [16]. However, the significance of astrocytes is underestimated in HIV neuropathogenesis even though only a small proportion of them are infected [17,18]. It is estimated that 20–57% of 84.6 billion ± 9.8 billion of nonneuronal cells may be astrocytes [19,20]. Importantly, astrocytes as well as microglia are long-lived populations [21–24].

HIV infection in astrocytes has been demonstrated in vivo by detection of viral DNA and RNA [17,18] or Nef expression in postmortem brain tissues from patients with AIDS [25]. Nevertheless, in-vitro studies show that only temporal or inefficient HIV infection occurs in cultures of astrocytes [26–28]. However, HIV pseudotyped with envelope of either murine leukemia virus or vesicular stomatitis virus (VSV-G) usually results in productive and long-lasting infection of astrocytes [29], indicating that HIV infection is mainly restricted at levels of viral entry or immediately after the entry and there is no significant intracellular obstacle to limit its replication [29–32]. This is further confirmed by other studies [33,34] although long-term HIV-1 latency can be established in a model of stem cell-derived astrocytes [35]. For entering a target cell, HIV needs to engage with CD4 and a co-receptor. Although CCR5-tropic (R5) viruses are important for viral transmission, the emergence of viruses that use CXCR4 (X4) or both co-receptors (R5X4) is associated with progression to AIDS [36]. Astrocytes lack CD4 expression [37] but express co-receptor CXCR4 [38,39] and CCR5 may be expressed under certain circumstances [39]. In this study, we explore HIV entry into astrocytes and propose a CD4 independent, CXCR4-dependent mechanism by which immature viral particles may evade lysosomal degradation and establish a productive infection.

Methods

Primary astrocytes

Human fetal astrocytes (HFA) were generated from human fetal brain specimens of 10–14 weeks’ gestation [40]. Mature astrocytes post five to six passages were used for experiments.

HIV stocks, viral infection and enhancement

HIV-1 infectious molecular clones and viral strains were obtained from NIH AIDS Reagent Program. HIV-1 NL4-3_based reporter virus clone, pNLENG1, was constructed by inserting EGFP gene with IRES between genes env and nef of pNL4-3 [41,42].

Viral stocks were produced by transfection in HEK293T cells or propagated in PBMCs or T-cell lines. For detecting integrated proviral DNA, DNA-free viral stocks were prepared by treatment with benzonase nuclease.

Astrocytes were infected with original virus stocks for 24 h, then washed and replenished with culture medium. The enhancement assays were performed by simultaneously treating the cells with 60–400 μmol/l chloroquine (ChQ) for 24 h and maintained in 20 μmol/l ChQ for up to 5 days.

Detection of proviral DNA

HFAs were pre-seeded in six-well plates, infected with DNA-free HIV stocks and simultaneously treated with or without ChQ. Detection of HIV-1 proviral DNA was designed and conducted based on Alu-PCR technique [43,44].

Transwell culture and infection-blocking assay

Either 2--3 × 104 HFAs or U373 MG-derived cells were pre-seeded in 24-well plates. 3--4 × 105 HIV-infected Jurkat-Tat (JKT) cells (4--5 days postinfection) were added to each of the Costar transwells with 0.4 μm pores (Corning, New York, USA). The inserts were removed after 3--6 days of culture and, the astrocytes in the plates were washed with PBS. The infection-blocking assay was performed as previously reported [40].

Detailed methods are described in the Supplemental Information, https://links.lww.com/QAD/B688.

Results

Persistent HIV infection can be established in primary astrocytes through multiple methods that overcome blockage on viral entry

To verify the ability of astrocytes to support HIV infection and replication, primary cultures of HFAs were transfected with full-length HIV-1 proviral DNA pYK-JRCSF or pNL4-3-based reporter construct pNENG1 (Fig. 1 a and Fig. 1S1A, upper panel. https://links.lww.com/QAD/B689) or infected with pseudoviruses (NL4-3 Y’/VSV-G) (Fig. 1 a and Fig. S1A lower panel, https://links.lww.com/QAD/B689). The infection with productive viral release persisted over the 2-month duration of the experiment. In contrast, HIV-1 p24 levels declined rapidly to undetectable levels within 2--3 weeks when HFAs were infected with cell-free HIV-1 NL4-3 or YK-JRCSF (Fig. 1 a). Furthermore, HIV infection could be easily established in HFAs that were pre-transfected with CD4 plasmid (Fig. 1 b and c and Fig. S1B and S1C, left, https://links.lww.com/QAD/B689), but no infection occurred in the cells transfected with empty plasmid (Fig. 1 b and c, and S1C, right, https://links.lww.com/QAD/B689). These observations confirm that there is no significant intracellular obstacle to block productive HIV infection in astrocytes, but major limitation is at level of viral entry.

F1
Fig. 1:
Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.
F2
Fig. 1 (continued):
Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.

Endocytosis has been proposed as a mechanism of HIV entry into astrocytes and the virus needs to escape endosomes/endolysosomes for establishing an infection [45–47]. To further confirm if HIV retention or degradation in lysosomes is a barrier to productive infection, a series of experiments were conducted. Initially, HFAs or progenitor-derived astrocytes (PDA) were simultaneously treated with a lysosomotropic agent, chloroquine (ChQ), while infected with X4-tropic, NL4-3_based reporter virus NLENG1. EGFP-expressing cells appeared in 3--5 days postinfection confirming HIV infection (Fig. 1 d) and persistently remained with some increase over 1 month (Fig. 1 e). Similar results were observed in HFAs infected with R5-tropic, SF162_based reporter virus SF162R3 (Fig. 1 f and g). However, no significant infection was detected in the cells without ChQ-treatment (Fig. 1 d--g). Importantly, the ChQ-mediated infection was clearly dose-dependent (Fig. 1 h) and persistent viral production remained stable up to 30 days postinfection (Fig. 1 i--k).

For clarification, sometimes low ‘infection’ might be detected in the cells without ChQ treatment. Extrachromosomal expression of HIV plasmids or artificial ‘entry’ of viral particles might be produced because of the facilitation of transfection reagents if transfection-based viral stocks were used for the infection (Fig. 1 i and j; and data not shown). However, this was not seen if plasmid-free viral stocks collected from HIV-infected cells was applied to the infection, such as IIIB (Fig. 1 k).

To further verify these observations, integrated proviral DNA was measured by Alu-PCR technique [43,44]. Results showed that integrated HIV DNA was significantly increased over time in the HFAs infected with DNA-free viral stocks IIIB or 92HT599 in presence of ChQ (Fig. 1 l and m), but it was very low and not increased in the infected cells without ChQ treatment (Fig. 1 l and m). This indicates that ‘non-productive/limited HIV infection’ [17] is not normally seen in the cultures of astrocytes.

To determine if chronically HIV-infected astrocytes could produce infectious viral particles, the HFAs initially generated with ChQ-mediated infection of NLENG1 were co-cultivated with lymphocytic cell lines. Viral transmission easily occurred when the 50 days (Fig. S1D i, ii, iii, https://links.lww.com/QAD/B689), 122 days (Fig. S1D iv, https://links.lww.com/QAD/B689) and 145 days (Fig. S1D v, https://links.lww.com/QAD/B689)-infected astrocytes were co-cultivated with JKT cells. The transmission also occurred when the 93 days-infected astrocytes were co-cultivated with Jurkat E6-1 cells (Fig. S1D vi, https://links.lww.com/QAD/B689).

HIV enters astrocytes via endocytosis, but requires escaping lysosomal degradation for establishing a productive infection

The process of HIV entry into astrocytes was further investigated by electron microscopy. A distinct phenomenon was observed, that the density significantly increased around the area of astrocyte membrane where viral particles attached to [Fig. 2 a(i--iii)]. This was followed by an invagination of the cell membrane [Fig. 2 a(iv)], and subsequent partial or complete endocytosis [Fig. 2 b(i--iv)]. In addition to this, a bridge-like connection was also noted between the viral particle and the cell membrane [Fig. 2 b(iv)]. These features suggest that HIV attachment to astrocytes should be receptor-mediated. Furthermore, HIV particles were observed to exist in the endosome [Fig. 2 c(i)] and endolysosome [Fig. 2 c(ii and iii)].

F3
Fig. 2:
HIV entry by endocytosis and enhanced infection with Tat-HA2 in primary astrocytes.
F4
Fig. 2 (Continued):
HIV entry by endocytosis and enhanced infection with Tat-HA2 in primary astrocytes.

As chloroquine has multiple effects on cellular function and is cytotoxic, we synthesized a Tat-HA2 fusion peptide containing HIV-1 Tat basic domain and influenza virus hemagglutinin subunit HA2 [48] to further investigate how retention of HIV in the compartments of endosomes/endolysosomes restricts infection of astrocytes (Fig. 2 d). Tat-basic peptide can bind to both HIV particles and cell membrane [49]. Hydrophobic N-terminal sequence of HA2 disrupts lipid membranes of low-pH cellular organelles (e.g. endosomes/lysosomes) [50]. HIV inocula were pre-incubated with Tat-HA2 or control peptides and infection assays were performed in astrocytes as mentioned above. Dose-dependent, enhanced infection was observed with NL4-3 (Fig. 2 e), NLENG1 (Fig. 2 f), YK-JRCSF (Fig. 2 g) and F162R3 (Fig. 2 h) in presence of Tat-HA2. However, enhanced infection was not detected with control peptides HA2, Tat_b (Tat basic peptide) and Tat_b+c (Tat peptide consisting of Tat basic and core domains [49]) (Fig. 2 e--h).

Detection of CD4 and coreceptors in astrocytes

A previous study showed that CD4 mRNA was detected in astrocytes despite no expression of CD4 protein [51]. Thus, we investigated if residual CD4 played a role in HIV infection of astrocytes. CD4 mRNA was measured in different sources of astrocytes via qPCR. Copy number of CD4 mRNA was over 6000 per 5 × 108 copies of β-actin mRNA in astrocytes from all sources (Fig. 3 a), whereas that of CXCR4 mRNA was over 2 × 105 per 1 × 108 copies of β- actin mRNA (Fig. 3 b). CCR5 and DC-Sign were not consistently detected in astrocytes (Fig. 3 c and d). Consistent with previous reports [37,51,52], CD4 was not detected in astrocytes by western blot or immunostaining. However, it was consistently detected in PDAs and HFAs by immunoprecipitation assay (Fig. 3 e). Even so, residual CD4 was not able to mediate HIV infection in astrocytes (Fig. 1 ).

F5
Fig. 3:
Low level of CD4 expression and regulation of CD4, CXCR4 mRNA by pro-inflammatory cytokines in primary astrocytes
F6
Fig. 3 (Continued):
Low level of CD4 expression and regulation of CD4, CXCR4 mRNA by pro-inflammatory cytokines in primary astrocytes

Pro-inflammatory cytokines up-regulate mRNA levels of CD4 and CXCR4 but has no effect on HIV infection in astrocytes

As HIV infection is restricted in astrocytes because of viral retention and degradation in endolysosomes, more efficient infection may occur by other mechanisms in vivo. It is well established that pro-inflammatory cytokines (e.g. IFN-γ, TNF-α, IL-1β, IL-6) are elevated in the CNS of patients with HIV-associated neurocognitive disorders (HAND) [53,54]. Hence, we treated HFAs with single or various combinations of cytokines and found that mRNA levels of both CD4 and CXCR4 were significantly increased 3 days posttreatment (Fig. 3 f and g). The up-regulation of CD4 mRNA was specifically observed following the treatments with IFN-γ alone, IFN-γ with TNF-α or IL-1β; an increase of nearly 60-fold was seen in the combination of three cytokines (Fig. 3 f). Interestingly, CXCR4 mRNA was down-regulated by IFN-γ alone in astrocytes, but most significantly upregulated by the combination of three cytokinndes (Fig. 3 g). These up-regulations are dose-dependent (Fig. S2A and S2B, https://links.lww.com/QAD/B689).

Yet, pre-treatments of HFAs with three cytokines (IFN-γ, TNF-α and IL-1β) had no effect on HIV infection as tested for multiple viral strains (Fig. 3 h and i). Consistent with this finding, no increase of CD4 protein was seen following treatment with cytokines (Fig. 3 j). Flow cytometry analysis further confirmed that percentage of CXCR4+ astrocytes was even slightly decreased and CD4 was still negative after the treatment of cytokines (Fig. 3 k).

Productive HIV infection in astrocytes is observed in a transwell culture system via a CD4-independent, CXCR4-dependent mechanism

Although cell-free HIV does not infect astrocytes or the infection is extremely low, cell-to-cell contact can significantly facilitate viral transmission from HIV-infected lymphocytes to astrocytes [55–58], whereby budding or immature HIV particles may productively infect astrocytes via a CXCR4-dependent, CD4-independent mechanism [40]. To test the hypothesis that immature HIV particles released from the infected lymphocytes may infect astrocytes in absence of cell-to-cell contact, we used a transwell culture system in which HIV-infected lymphocytes were loaded in the upper transwells and HFAs were seeded in culture plate (Fig. 4 a). This prevented astrocytes from directly contacting with HIV-infected lymphocytes but allowed viral particles to cross through the pores (0.4 μm) of transwell membrane (Fig. 4 a).

F7
Fig. 4:
Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.
F8
Fig. 4 (Continued):
Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.

Using this system, HIV could consistently infect astrocytes (Fig. 4 b); after removal of the transwells, the viruses were constantly released from NLENG1-infected astrocytes into culture media (Fig. 4 c). Importantly, the infection in astrocytes could be significantly blocked by anti-CXCR4 antibody or its antagonist AMD3100 but was only partially inhibited by anti-CD4 antibody or fusion inhibitor T20 (Fig. 4 c and d); anti-DC-SIGN antibody had no effect (Fig. 4 c and d). The infection by this system was further observed with other HIV-1 strains, such as 89.6 (R5X4), 93US151 (R5X4), 92HT599 (X4) (Fig. 4 e). Integrated proviral DNA in the infected HFAs increased over time (Fig. 4 f).

To further explore the CD4-independent, CXCR4-dependent mechanism of HIV infection via the transwell culture, we generated a panel of cell lines from U373 MG cells by knocking out CD4 and/or CXCR4 via CRISPR/Cas9 technique, including CD4− CXCR4− U373 MG_1-6, CD4+ CXCR4− U373 MG_1-8, CD4−CXCR4+ U373 MG_2-4 and CD4+CXCR4+ U373 MG_2-10 (Fig. 4 g). Next, these cell lines were infected in parallel by the transwell cultures loaded with NLENG1-infected JKTs or with cell-free viral supernatants. We found that U373 MG_2-10 cells were significantly infected by both cell-free HIV and the transwell cultures (Fig. 4 h). However, U373 MG_2-4 cells were only infected in the transwell cultures but not with cell-free virus HIV (Fig. 4 i). No significant infection was observed in U373 MG_1-6 and U373 MG_1-8 cells with either of them (Fig. 4 j and k). HIV-1 p24 declined rapidly in the supernatants from cell-free HIV-infected U373 MG_1-6, U373 MG_1-8 and U373 MG_2-4 cells (Fig. 4 j). Nevertheless, it persistently remained over 200 pg/ml during 3 weeks of culture in U373 MG_2-4, in which the infection was established via the transwell cultures (Fig. 4 k). The ratio of p24 values comparing the transwell cultures to cell-free HIV infections increased over time and was maximally greater than 15 in U373 MG_2-4, but it remained around 1 in U373 MG_1-6 and U373 MG_1-8 (Fig. 4 l). These data indicate that CXCR4 plays an essential role for establishing a productive HIV infection in U373 MG_2-4 via the transwell cultures, comparable with the findings in primary astrocytes.

Discussion

HIV infection of astrocytes in the brain has been established as an important viral reservoir by cumulative in-vivo evidence [59–64]; and even though productive infection was reported [65–68], the prevailing opinion has been that the infection might be nonproductive or restricted [17,60,69–72]. This is in part because infection has been difficult to accomplish in vitro when infection assays are performed with cell-free HIV [32,33,45,73]. Hence, we systemically investigated if the barrier to HIV infection was at viral entry or postentry and determined mechanisms by which the barrier could be overcome to establish a productive infection.

Previous studies have shown that there was a significant barrier for HIV entry or intracellular restrictions in astrocytes or astroglioma cell lines [29,34,45,74–77]. We confirmed that a similar barrier existed in primary astrocytes that could be completely overcome by transfection of full-length HIV proviral DNA, infection with VSV-G/HIV pseudoviruses or by transfection with CD4-expressing plasmid followed infection with HIV. As CD4 is not detected in astrocytes [37,52], the barrier to HIV entry can be attributed to absence of CD4. A number of proteins have been identified on the cell membrane of astrocytes that bind to HIV envelope, gp120, such as galactosyl ceramide [78,79], mannose receptor [46], chemokine receptor D6 [80] and other proteins [52,81]. However, these receptors either do not or only minimally mediate HIV entry into astrocytes [46,82]. Previous studies have demonstrated that HIV could enter astrocytes by endocytosis [45,46,77]. In the current study, we further provided evidence demonstrating that the process of HIV entry by endocytosis only resulted in an abortive infection in astrocytes. Highly increased density at the sites of the cell membrane where viral particles attached during HIV internalization is suggestive of a receptor-mediated endocytosis (Fig. 2 ), likely mediated by one of the receptors described above. The endocytosis could not lead to HIV infection or proviral DNA integration; viral particles got trapped in the endosomes/endolysosomes where they were degraded [32,47]. However, the retention of endocytosed HIV in the endosomal/lysosomal compartments could be disrupted by treatment with lysosomotrophic agent, such as chloroquine or Tat-HA2 peptide (Figs. 1 and 2), which facilitated HIV lysosomal escape and release of viral RNA into the cytosol leading to a productive infection with proviral DNA integration [32,47]. Therefore, in normal condition, HIV is unable to establish a productive or nonproductive infection in astrocytes; but once infected, astrocytes can persistently release the viruses and serve as a long-term reservoir.

Previous studies have not detected CD4 protein in astrocytes even though CD4 mRNA was expressed [51]. Although CD4 was not detectable by immunostaining or western blot analysis, we consistently detected CD4 mRNA by qPCR and minimal levels of CD4 protein by immunoprecipitation in astrocytes from different sources. However, even combinations of pro-inflammatory cytokines (IL-1β, IFN-γ, TNF-α) [54,83] that were able to markedly increase transcripts of CD4 and CXCR4 [39,84] did not result in increased proteins or enhancement of HIV infection. This suggests that there is either a translational block of these proteins or posttranslational degradation in astrocytes, probably associated with these cytokines.

We and others have previously shown that HIV infection of astrocytes could be established by cell-to-cell contact [40,55–58], and under that condition, immature HIV particles directly budded onto the membrane of astrocytes and might infect astrocytes via a CD4-independent, CXCR4-dependent mechanism [40]. In the current study, we showed that cell-to-cell contact might not be essential for this transmission to occur. Using the transwell culture system, we demonstrated that close proximity of HIV-infected lymphocytes to astrocytes was sufficient for the CD4-independent, CXCR4-dependent infection to occur. This was supported by a series of experiments in which CXCR4 antibody and antagonist significantly blocked the infection and it only occurred by X4 and R5X4 viruses but not by R5 viruses [33]. To further confirm the role of CXCR4, we created cell lines that exclusively expressed either CD4, CXCR4 or both, and found that using the transwell cultures, expression of CXCR4 alone was sufficient for the infection to occur.

On the basis of a series of observations described above, a model of HIV infection in astrocytes is proposed as follows: cell-free, mature virus attaches to astrocyte via an unspecified receptor that results in endocytosis (Fig. 5a), and the virus is retained in the endosome/endolysosome and gets degraded (Fig. 5a) [32]; an immature viral particle (a transient form of HIV virion) released from an HIV-infected lymphocyte passes through the pores of transwell membrane and reach an astrocyte to initiate the cycle of infection via the CD4-independent, CXCR4-dependent manner before the process of viral maturation is completed (Fig. 5b) as described previously [40]. In a typical life cycle, HIV binds to CD4 inducing a conformational change in gp120 that exposes specific epitopes for binding to CXCR4 [85]; however, cell-free HIV stocks normally only contain mature viral particles, which are incapable of infecting astrocytes in absence of CD4. In the context of cell-to-cell contact or close proximity with HIV-infected cells, CXCR4 alone is sufficient to make the infection to occur, indicating that some of viral particles may directly bind to CXCR4 and fuse with the astrocyte membrane. Under these circumstances, immature viral particles are the best candidate. We hypothesize that gp120 on immature HIV virion might have exposed CXCR4-binding sites and could directly bind to CXCR4 on astrocyte to induce membrane fusion leading to a productive HIV infection [40] (Fig. 5b). As HIV maturation occurs after their release from the infected cells [86], immature viral particles as they are budding off would have a chance to bind to CXCR4 in this transition period (Fig. 5b). However, they might not be able to immediately trigger the fusion process before undergoing the proteolytic maturation to form a functional core because of the absence of infectivity at the immature stage [87–90]. This model explains why cell-to-cell contact or close proximity to HIV-infected lymphocytes is capable of triggering HIV infection in astrocytes as it represents the best opportunity for exposure to immature viral particles, although other possibilities cannot be excluded and the model may not fully represent the situation in vivo. Interestingly, a CD4-independent, CXCR4-dependent viral entry is also described in HIV-2 even though its mechanism has not been studied yet [91,92].

F9
Fig. 5:
Model of HIV infection in human astrocytes.

These findings are highly relevant to HIV neuropathogenesis. Our observations provide a mechanism that explains why astrocytes are infected predominantly in the perivascular regions [18]. The brain vasculature is unique, where foot processes of astrocytes are in contact with endothelial cells, macrophage or microglia, and are in the integral part of blood--brain barrier. Thus, as HIV-infected lymphocytes traffic through the blood--brain barrier, immature viral particles are released and might infect perivascular astrocytes. Similarly, immature R5X4 viruses released from HIV-infected macrophage or microglia might also infect astrocytes in the perivascular regions. Thus, CXCR4 inhibitors may help prevent the formation of HIV reservoir in astrocytes. It also raises a concern that the use of CCR5 antagonists may drive emergence of X4 viruses [93] and increase HIV reservoir of astrocytes within the brain.

Acknowledgements

We thank Dr David Levy at New York University School of Dentistry for providing HIV-1 reporter pNLENG1; Dr Amanda Brown at Johns Hopkins University (JHU) for providing pSF162 and pSF162R3; Michael Delannoy at JHU for technical assistance with electron microscopy; Dr Ashok Chauhan at University of South Carolina School of Medicine for providing NL4-3Y’/VSV-G pseudoviruses; and Alan Hoofring at NIH Medical Arts for drawing diagrams. We also thank the following colleagues at NINDS, NIH: Dr Maria Chiara Monaco for making PDAs; Drs. Joseph Steiner and Alina Popescu for assistance with experiments; Dr Lisa Henderson for careful reading of the manuscript and helpful comments; Dr Tianxia Wu for statistical analysis.

Conflicts of interest

There are no conflicts of interest.

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Keywords:

astrocyte; CXCR4; cytokine; endocytosis; HIV; integration; reservoir

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