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Vol.:(0123456789)
Journal of NeuroVirology
https://doi.org/10.1007/s13365-024-01207-w
Characterization ofHIV variants frompaired Cerebrospinal fluid
andPlasma samples inprimary microglia and CD4+ T‑cells
StephanieB.H.Gumbs1· ArjenJ.Stam1,2· TaniaMudrikova2· PaulineJ.Schipper1· AndyI.M.Hoepelman2·
PetraM.vanHam1· AnneL.Borst1· LMarijeHofstra1· LavinaGharu1· StephanievanWyk3· EduanWilkinson4·
LotD.deWitte5· AnnemarieM.J.Wensing1 · MoniqueNijhuis1
Received: 25 June 2023 / Revised: 30 March 2024 / Accepted: 3 April 2024
© The Author(s) 2024
Abstract
Despite antiretroviral therapy (ART), HIV persistence in the central nervous system (CNS) continues to cause a range of cog-
nitive impairments in people living with HIV (PLWH). Upon disease progression, transmigrating CCR5-using T-cell tropic
viruses are hypothesized to evolve into macrophage-tropic viruses in the CNS that can efficiently infect low CD4-expressing
cells, such as microglia. We examined HIV-1 RNA concentration, co-receptor usage, and CSF compartmentalization in paired
CSF and blood samples from 19 adults not on treatment. Full-length envelope CSF- and plasma-derived reporter viruses
were generated from 3 subjects and phenotypically characterized in human primary CD4+ T-cells and primary microglia.
Median HIV RNA levels were higher in plasma than in CSF (5.01 vs. 4.12 log10 cp/mL; p = 0.004), and coreceptor usage
was mostly concordant for CCR5 across the paired samples (n = 17). Genetically compartmentalized CSF viral populations
were detected in 2 subjects, one with and one without neurological symptoms. All viral clones could replicate in T-cells (R5
T cell-tropic). In addition, 3 CSF and 1 plasma patient-derived viral clones also had the capacity to replicate in microglia/
macrophages and, therefore have an intermediate macrophage tropic phenotype. Overall, with this study, we demonstrate
that in a subset of PLWH, plasma-derived viruses undergo genetic and phenotypic evolution within the CNS, indicating
viral infection and replication in CNS cells. It remains to be studied whether the intermediate macrophage-tropic phenotype
observed in primary microglia represents a midpoint in the evolution towards a macrophage-tropic phenotype that can effi-
ciently replicate in microglial cells and propagate viral infection in the CNS.
Keywords HIV· CSF· Plasma· Compartmentalization· Microglia
Introduction
Despite antiretroviral therapy (ART), HIV persistence in the
central nervous system (CNS) continues to affect a large
portion of people living with HIV (PLWH), resulting in a
Stephanie B. H. Gumbs and Arjen J. Stam contributed equally to
this work.
Annemarie M. J. Wensing and Monique Nijhuis share last co-
authorship.
* Annemarie M. J. Wensing
a.m.j.wensing@umcutrecht.nl
1 Translational Virology, Department ofMedical
Microbiology, University Medical Center Utrecht,
3584CXUtrecht, TheNetherlands
2 Department ofInternal Medicine andInfectious Diseases,
University Medical Center Utrecht, 3584CXUtrecht,
TheNetherlands
3 Centre forEpidemic Response andInnovation (CERI),
School ofData Science andComputational Thinking,
Stellenbosch University, Stellenbosch, SouthAfrica
4 KwaZulu-Natal Research Innovation andSequencing
Platform (KRISP), School ofLaboratory Medicine
andMedical Sciences, University ofKwaZulu-Natal,
Durban, SouthAfrica
5 Department ofPsychiatry, Icahn School ofMedicine
atMount Sinai, NewYork, NY10029, USA
Journal of NeuroVirology
wide range of cognitive impairments (Heaton etal. 2010).
The onset and progression of HIV-associated neurocognitive
disorder (HAND) is believed to be multifactorial, includ-
ing continued immune dysregulation and residual chronic
inflammation in response to low-level virus production (or
replication) and cytotoxic viral proteins (Clifford and Ances
2013; Jadhav and Nema 2021).
During early infection, R5 T-cell tropic viruses, charac-
terized by their ability to efficiently enter CD4+ T-cells but
not macrophages and microglia, represent the majority of
the viral population (Joseph and Swanstrom 2018). Upon
disease progression, genetically distinct viral populations
can be found in cerebrospinal fluid (CSF) and brain tissue of
both untreated and virally suppressed individuals, irrespec-
tive of the presence of neurological disorders (Bednar etal.
2015; Borrajo etal. 2021; Chan and Spudich 2022). Exten-
sive research has been conducted on the genetic compart-
mentalization between the CNS and blood, however their
phenotypic characteristics remain poorly understood. R5
T-cell tropic viruses are predominant in both compartments.
In general R5 plasma derived viruses are T cell tropic, while
CNS derived viruses, in addition to T cell tropic virus, can
also harbor viruses that are M-tropic, referring to their
enhanced ability to infect cells with low CD4 surface expres-
sion such as macrophages and microglia (Brese etal. 2018;
Gonzalez-Perez etal. 2012; Schnell etal. 2011; Sturdevant
etal. 2012). Accordingly, HIV DNA and/or RNA within
the CNS are mostly found in perivascular macrophages and
microglia (Ko etal. 2019; Lamers etal. 2016; Tso etal.
2018).
Previous studies have examined the CD4 entry phenotype
of CNS- and plasma-derived pseudotyped viruses using the
Affinofile cell line, on which CD4 and CCR5 surface expres-
sion can be differentially induced, and monocyte-derived
macrophages (Arrildt etal. 2015; Joseph etal. 2014; Schnell
etal. 2011). While the Affinofile cell line is commonly used
for entry tropism analysis, this model system is derived from
a T-cell line and therefore cannot fully represent the entry
determinants for primary microglia, such as attachment
receptors, endocytosis mechanisms, and microglia-specific
restriction factors. Therefore, it remains to be determined
whether M-tropic HIV variants have the same entry advan-
tage for microglia as they do for low CD4 Affinofile cells and
monocyte-derived macrophages. In this study, we examined
potential genetic compartmentalization between paired CSF-
and plasma-derived HIV variants and gain more insight into
their entry affinity for human primary CD4+ T-cells and pri-
mary microglia. Paired CSF- and plasma-derived HIV vari-
ants were isolated from viremic PLWH without antiretroviral
treatment and characterized based on coreceptor-usage and
genetic compartmentalization, followed by a phenotypical
analysis in CD4+ T-cells and microglia. To our knowledge,
this is the first study to combine genetic characterization
with a phenotypical analysis in human primary blood and
CNS cells.
Methods
Design andstudy population
For this cross-sectional study, paired CSF-plasma samples
were collected from stored samples in the period 2001–2016
from patients in care at the University Medical Center Utre-
cht and participating in the Dutch ATHENA observational
cohort. Paired samples were obtained for clinical diagnos-
tic purposes [TableS1]. A total of 28 subjects, with and
without neurological symptoms, had sufficient material for
virological analysis, of which 9 were excluded due to being
on different antiretroviral therapies at the time of sampling.
CSF in neurosymptomatic subjects was collected for clini-
cal diagnostics purposes. The CSF of neuroasymptomatic
patients was primarily collected to exclude neurosyphilis, as
part of a standard clinical procedure in patients with TPHA
(treponema pallidum hemagglutination assay) or (previ-
ous) VDRL- positive plasma syphilis serology and in two
instances for a diagnostic work-up not including neurologi-
cal symptoms [S1]. Paired samples were defined as CSF and
plasma-EDTA (or serum) obtained within 7days from each
other. All patients had detectable HIV RNA in plasma at
the time of lumbar puncture and were ART-naïve or off-
treatment at the time of sampling [Tables1 and S1].
HIV RNA andplasma CD4 count analysis
HIV RNA levels in plasma and CSF were determined by an
ultrasensitive viral load assay with a reported cut-off value
of 50 copies/ml (Ampliprep/COBAS Taqman HIV-1 assay,
Roche).
Next‑generation sequencing andco‑receptor
prediction
Viral RNA was isolated according to the method devel-
oped by (Boom etal. 1990). The HIV-1 envelope V3 region
was amplified by RT-PCR (Titan One Tube RT-PCR kit,
Roche) followed by a nested PCR (Expand High Fidelity
PCR System, Roche), according to the manufacturer’s pro-
tocol. Please refer to supplementary TableS2 for a list of the
primers used. PCR products were purified with the Qiaquick
PCR purification kit (Qiagen). Library preparation was done
using a Nextera-XT DNA Library Preparation and Index kit
(Illumina, USA) according to the manufacturer’s instruc-
tions. The resulting libraries were normalized and pooled.
Sequencing was performed on an Illumina MiSeq plat-
form using the MiSeq Reagent Kit v2 for 500 cycles. After
Journal of NeuroVirology
Table 1 Clinical, virologic and phylogenetic characteristics of the subject population
N/A.: V3 sequences clonal in CSF or identical between CSF and plasma
a HIV disease stage according to the CDC 1993 Revised Classification System for HIV Infection (PMID: 1361652)
b estimated plasma CD4 cells/μL, value determined by test value closest to sampling time of pair
c NS neurosymptomatic, NA neuroasymptomatic
d VL viral load; estimated HIV-RNA (log10 copies/ml) determined by test value closest to sampling time of pair
e FPR False-positive rate; lowest and highest FPR detected in CSF and plasma based on V3 amplicons (Miseq sequencing) with the geno2pheno
algorithm
f Geno2pheno coreceptor prediction; R5: virus predicted to use the CCR5 co-receptor. X4: virus predicted to use the CXCR4 co-receptor
g Comparative genetic analyses of viral populations in blood plasma and cerebrospinal fluid using three statistical analyses: Wright’s measure of
population subdivision (Fst), Nearest-neighbor statistic (Snn) and the Slatkin-Maddison test (SM). Genetic compartmentalization was statisti-
cally significant at P values < 0.01
Analysis of compartmentaliza-
tiong
Subject ID Disease
state
(CDC)a
CD4bNS/NAcOrigin HIV VLdFPR RangeeTropismfFst Snn SM CSF Compart.h
1 A2 209 NA CSF 3.95 30.1—73.3 R5 0.20 0.02 0.302 EQ
Plasma 5.65 30.1—37.1 R5
2 A2 373 NA CSF 3.21 17.3 R5 N/A N/A N/A N/A
Plasma 5.01 17.3—46.8 R5
3 unknown 549 NA CSF 4.72 76.2—95.2 R5 0.66 0.91 1 EQ
Plasma 5.88 68.6—95.2 R5
4 unknown 400 NA CSF 3.13 12 R5 N/A N/A N/A N/A
Plasma 3.15 12—33.7 R5
6 C3 16 NS CSF 4.88 100 R5 0.15 < 0.0001 0.09 EQ
Plasma 5.34 4.8—99.2 R5
7 B2 246 NA CSF 3.72 26.2—78.1 R5 0.70 0.38 0.33 EQ
Plasma 4.31 48.7—72.1 R5
8 unknown 10 NS CSF 5.28 7.8—44.2 R5 0.33 0.67 0.26 EQ
Plasma 5.35 4—81 R5
10 A1 705 NA CSF 4.43 83—97 R5 0.84 0.80 0.24 EQ
Plasma 5.10 83—97 R5
12 A2 375 NA CSF 4.72 6.3—20.4 R5 0.21 0.59 0.1 EQ
Plasma 4.49 10.5—20.4 R5
13 unknown 30 NS CSF 5.21 74.6—91.2 R5 < 0.0001 < 0.0001 0.01 CP
Plasma 6.04 35.1—53.7 R5
14 A2 469 NS CSF 3.54 71.1 R5 0.54 0.34 0.59 EQ
Plasma 4.27 71.1—90.7 R5
16 C3 83 NS CSF 4.12 72—94.6 R5 0.72 0.86 0.6 EQ
Plasma 5.10 76—94.6 R5
17 A1 424 NS CSF 4.38 2.5—5 X4/R5 0.70 0.01 0.04 EQ
Plasma 3.17 1.7—55.1 X4/R5
18 A1 574 NS CSF 2.87 15—46.8 R5 0.44 0.33 0.13 EQ
Plasma 4.56 15—46.8 R5
19 A1 387 NA CSF 4.77 16—87 R5 < 0.0001 < 0.0001 < 0.0001 CP
Plasma 4.18 0.2—90.3 X4/R5
20 A0 139 NA CSF 3.25 64—70.8 R5 0.27 0.53 1 EQ
Plasma 3.71 56.1—74.4 R5
21 A2 399 NS CSF 3.74 30.1—52.1 R5 0.29 0.92 0.16 EQ
Plasma 3.78 30.1—52.1 R5
25 A0 362 NS CSF 3.06 25.2 R5 N/A N/A N/A N/A
Plasma 5.88 25.2 R5
27 C3 185 NS CSF 4.95 38.8—86.2 R5 0.56 0.73 0.38 EQ
Plasma 5.73 8.5—86.2 R5
Journal of NeuroVirology
aligning the sequence reads of each subject with the consen-
sus sequence of their respective subtype, reads that overlap
the entire V3 region were isolated and trimmed. Unique V3
sequences with a prevalence of > 1% in the population were
used for HIV-1 co-receptor tropism. Co-receptor usage was
predicted with the Geno2pheno[coreceptor] algorithm ver-
sion 2.5 for deep sequences with the recommended false-
positive rate (FPR) cut-off value for deep V3 sequencing of
3.5% (Swenson etal. 2011). It is predicted that an FPR value
below 3.5% indicates an X4-tropic virus, whereas a value
above 3.5% indicates an R5-tropic virus.
Compartmentalization analysis
Genetic compartmentalization between the CSF and plasma-
derived viral population, was determined for each subject
based on only deep-sequenced V3 sequences using three
methods: Wright’s measure of population subdivision
(Fst) (Hudson etal. 1992), Nearest-neighbor statistic (Snn)
(Hudson 2000), and the tree-based Slatkin-Maddison test
(SM) (Slatkin and Maddison 1989). Wright’s measure of
population subdivision (Fst) quantifies the genetic variance
between populations relative to the total genetic variance.
Higher Fst values indicate greater population differentia-
tion. The nearest-neighbor statistic (Snn) assesses how often
sequences' nearest neighbors are from the same compart-
ment, with values closer to 1 indicating stronger compart-
mentalization. The Slatkin-Maddison (SM) test evaluates
the number of migrations needed to explain the distribution
of lineages between populations, with fewer migrations sug-
gesting compartmentalization. All three methods were con-
ducted with the HyPhy software version 2.2.4 (Kosakovsky
Pond etal. 2005). For the two distance-based methods (Fst
and Snn), the Tamura-Nei 93 algorithm was applied along
with a bootstrap value and permutation test of 10,000 with
only deep-sequenced V3 sequences as input. For the tree-
based SM test, multiple sequence alignments of the HIV
env V3 regions were performed using the online version of
the MAFFT software (https:// mafft. cbrc. jp/ align ment/ server/
index. html) (Katoh etal. 2018), accessed 2022/08/09 using
standard parameters for nucleic acid alignment. A Nearest
Neighbor Joining Tree was constructed using MEGA version
11.0.11(Tamura etal. 2021), and the Kimura 2-parameter
substitution model was applied. Compartmentalization was
evaluated using the nearest-neighbor statistic (Snn) (Hudson
2000), applying 10 000 permutations, implemented using the
HyPhy software package version 2.2.4 (Kosakovsky Pond
etal. 2005). CSF viral populations were defined as either
compartmentalized (cp), if all three tests were significant
(p < 0.01), or equilibrated (eq) if statistical significance was
not reached (p > 0.01) in one or more of these tests (Zárate
etal. 2007).
Generation ofHIV viral clones
The HIV-1 envelope (gp160) region was amplified by RT-
PCR (Superscript IV Reverse Transcriptase Kit, Invitrogen)
followed by a nested PCR (Platinum Taq Superfi PCR Mas-
ter Mix, Invitrogen), according to the manufacturer’s proto-
col. Please refer to supplementary TableS2 for a list of the
primers used. Envelope amplicons were introduced into a
HxB2 gp160deletion vector with a luciferase reporter gene
(HxB2ΔENVluc), previously described in (Gumbs etal.
2022), using the NEBuilder HiFi DNA Assembly Cloning
Kit (New England Biolabs). We used the same vector car-
rying either the gp160 sequence of JRCSF (R5 T-tropic),
YU-2 (R5 M-tropic) or BaL (R5-tropic) as controls. Hek-
293T cells were transfected with the chimeric plasmids
using lipofectamine 2000 reagent (Invitrogen). After 48h,
the supernatant containing replication-competent virus was
harvested and stored at −80°C until further use. p24 was
determined with an ELISA p24 assay (Aalto Bioreagent,
Dublin, Ireland).
HIV Infection inprimary microglia and CD4+ T‑cells
Fresh postmortem adult human brain tissue was provided
by the Netherlands Brain Bank (NBB). The isolation of
primary microglia was conducted according to the proto-
col described previously with some minor modifications for
human brain tissue (Mattei etal. 2020). Following isolation,
primary microglia were cultured in poly-L-lysine hydro-
bromide (PLL)-coated 96-well plates (1 × 105cells/well) in
microglia medium (RPMI 1640 (Gibco Life Technologies)
supplemented with 10% FCS, 1% penicillin–streptomycin
(Gibco Life Technologies) and 100ng/mL IL-34 (Miltenyi
Biotec)) for 2–3days before infection. Primary microglia
were infected overnight with 10ng (p24Gag) virus after
which the medium was fully replaced and cells were cul-
tured up to 17days in microglia medium without medium
refreshment.
PBMC were isolated from peripheral blood obtained
from healthy donors by Ficoll-Paque density gradient.
CD4+ T-cells were subsequently isolated through negative
selection with the CD4+ T Cell Isolation Kit (Miltenyi Bio-
tec 130–096-533), according to the manufacturer’s protocol.
h Characteristics of the HIV viral population in the CSF compartment (compart): CP (compartmentalized), if all three tests were significant
(p < 0.01), or equilibrated (EQ) if statistical significance was not reached (p > 0.01)
Table 1 (continued)
Journal of NeuroVirology
Before infection, CD4+ T-cells were stimulated for 2days
in culture medium (RPMI 1640 (Gibco Life Technologies)
with 10% Fetal Bovine Serum, 1% penicillin–streptomycin
(Gibco Life Technologies) and IL-2 (20U/mL) (Invitrogen))
supplemented with Phytohaemagglutinin (5µg/mL). CD4
infection was performed in Duplo for 3h with 10ng (p24
Gag) virus per 100.000 cells in Eppendorf’s placed on a
tube rotator. For the Maraviroc experiment, CD4+ T-cells
were treated with 100nM MVC for 1h before infection.
Following infection, medium was fully replaced with cul-
ture medium and CD4+ T-cells were cultured for 14days
in 96-well plates without medium refreshment.
Luminescence
Supernatant was collected 2–3 times per week and lumi-
nescence was measured according to the manufacturer’s
protocol with the Nano-Glo® Luciferase Assay System
(Promega). The graphs were created with GraphPad Prism
version 8.3.0.
Statistical analysis
All data were analyzed with GraphPad Prism version 8.3.0.
Descriptive statistics were used to compare the characteris-
tics between the paired plasma and CSF samples. The non-
parametric Wilcoxon signed rank test is used to compare
groups with paired data, including the differences in FPR.
Differences within continuous variables (e.g., viral load)
compared to categorical data (e.g., neurological symptoms)
were performed by using the Mann–Whitney U test. A Pear-
son correlation is used to determine the association of HIV-
RNA levels.
Results
Clinical characteristics
Paired plasma and CSF samples were collected from 19
adult subjects enrolled in the Dutch ATHENA observational
cohort subjects were mainly infected with Subtype B virus,
except for subject 6 (subtype CRF02_AG) and subject 25
(CRF12_BF), and were ART naïve or off treatment at the
time of sampling for at least 1month. The majority of the
study population was male, with a mean age of 47years and
a mean CD4+ T-cell count of 312 cells/µL. The clinical and
virological characteristics of each subject can be found in
Table1 and TableS1.
Median HIV RNA levels were significantly higher in
plasma than in CSF (5.01 vs. 4.12 log10 cp/mL; p = 0.004)
and moderately correlated with each other (Pearson r = 0.42;
p = 0.04) [Fig.1a, b]. A similar pattern in virus concentration
was also observed in the neurosymptomatic subjects (5.22
vs. 4.25 log10 cp/mL; p = 0.05) [Fig.1d]. However, further
research with a larger sample size is needed to determine
whether this finding is also true for neuroasymptomatic sub-
jects [Fig.1c]. A comparison of the CSF and plasma RNA
levels between the neurosymptomatic and neuroasympto-
matic subjects, however, showed no significant difference
[Fig.1e, f].
Analyses ofCNS compartmentalization
Genetic compartmentalization between CSF and plasma
HIV variants was determined based on the V3 region in
the viral envelope (env) gene. CSF viral populations were
defined as either compartmentalized (cp), if all three com-
partmentalization analyses (Fst, Snn, SM) were significant
(p < 0.01), or equilibrated (eq) if statistical significance was
not reached (p > 0.01) (Zárate etal. 2007) [Table1]. Most
of the subjects (89%) had equilibrated viral populations in
their CSF and plasma. Significant genetic CNS compart-
mentalization was detected in two subjects, 13 and 19. Sub-
ject 13 had advanced disease (CD4 count 30 cells/μl) and
neurological symptoms consisting of balance disturbances
and peripheral weakness. In contrast, subject 19 had less
advanced HIV infection (CD4 count 387 cells/μl) and had no
neurological symptoms, suggesting that CNS compartmen-
talization does not always present with clinically observable
neurological symptoms.
CSF‑derived viral variants can efficiently enter
CD4+ T‑cells, withamodest enhancement forviral
entry inlow‑CD4 expressing primary microglia
Co-receptor usage was mostly concordant across the paired
samples, with 17 out of 19 pairs predicted to consist exclu-
sively of CCR5-using viral strains in both compartments
[Table1]. A comparison of the lowest FPR values in plasma
and CSF revealed no correlation or significant difference
[Data not shown]. Subject 17 is predicted to have CCR5- and
CXCR4-using viral strains in both plasma and CSF, whereas
subject 19 is predicted to have CXCR4- and CCR5-using
viruses in the plasma but only CCR5-using virus in the
CSF. In addition to being the only subject with CXCR4-
using virus in the CSF, subject 17 was also the only subject
diagnosed with severe symptoms (HIV encephalopathy) and
one of three subjects with a CSF HIV RNA load higher than
in plasma (difference 1.21 log10 copies/mL).
Within the CNS, HIV DNA is primarily detected in
perivascular macrophages and primary microglia (Joseph
etal. 2015). We investigated the entry phenotype of the CSF
and plasma HIV variants from compartmentalized (cp) neu-
roasymptomatic subject 19 and two equilibrated (eq) subjects,
subjects 8 and 27, with neurological symptoms [TableS1].
Journal of NeuroVirology
These three subjects were selected in a step-wise process. First,
we selected subjects with a difference in FPR value between
plasma and CSF, then we selected subjects from whom the
volume of stored CSF and plasma was sufficient for the exper-
iment and finally the clones needed to be viable from both
plasma and CSF. First, we generated CSF- and plasma-derived
luciferase reporter viruses using the full-length envelope (Env)
gene amplified from the CSF and the plasma of these three
subjects. For each subject, we obtained a diverse mixture of
viral clones with different FPR values between the CSF and
Fig. 1 Relationship of HIV-1
RNA levels (log 10cp/mL)
measured in paired plasma and
CSF samples in neuroasymp-
tomatic and neurosymptomatic
subjects. a Plots depicts the
correlation between HIV RNA
CSF and HIV RNA plasma for
all subjects. Black dashed line
represents line of identity. b,
c, d Boxes depict median HIV
RNA levels and IQR, measured
in plasma (red) and CSF (blue)
in all patients, neuroasympto-
matic and neurosymptomatic
subjects. e, f Boxes depict CSF
(blue) and plasma (red) median
HIV RNA levels and IQR
between neuroasymptomatic
and neurosymptomatic subjects.
* = Statistically significant
(p < 0.05)
0
2
4
6
8
Neuroasymptomatic
HIV-RNA(log)
Plasma CSF
0
2
4
6
8
Neurosymptomatic
HIV-RNA(log)
*
Plasma CSF
0
2
4
6
8CSF
HIV-RNA(log)
Neuro-
asymptomatic
Neuro-
symptomatic
0
2
4
6
8Plasma
HIV-RNA(log)
Neuro-
asymptomatic
Neuro-
sy
mptomatic
123456
1
2
3
4
5
6
HIV-RNA plasma (log)
HIV-RNA CSF(log)
Neuroasymptomatic
Neurosymptomatic
Plasma CSF
0
2
4
6
8
HIV-RNA(log)
*
ab
c
d
e
f
Journal of NeuroVirology
plasma [Fig.2]. These viral clones were phenotypically char-
acterized for viral entry into CD4+ T-cells (high CD4 surface
levels) and primary microglia (low CD4 surface levels), the
main HIV target cells in the blood and CNS. Considering that
CD4 surface expression levels on CD4+ T-cells and micro-
glia are likely to differ between donors, we used 3 different
donors and three laboratory strains as a control for infection:
two R5 M-tropic virus (Bal and YU-2) and one R5 T-tropic
virus (JRCSF).
For subjects 19 (cp) and 27 (eq), we observed no signifi-
cant difference between the ability of the CSF and plasma
viruses to infect CD4+ T-cells [Fig.2b, c]. In subject 8 (eq),
we observed a ≥ 10-fold higher infectivity with CSF clones 4
and 6, compared to the plasma viruses [Fig.2a]. Interestingly,
this infectivity was also substantially higher than CSF clone 1
which had the same FPR value, suggesting that there are other
determinants outside of the V3 loop that can greatly affect cell
entry. Furthermore, treatment with the CCR5 inhibitor maravi-
roc (MVC), supported co-receptor prediction and significantly
inhibited T-cell infection by the R5-predicted CSF and plasma
viruses, whereas the X4-predicted plasma viruses of subject
19 were resistant to MVC inhibition [Fig.2d–f]. R5-predicted
CSF clone 4 of subject 8, despite having a high FPR value of
28.8, was also greatly resistant to MVC inhibition (≥ 60%),
suggesting that this clone is dual tropic [Fig.2d].
Lastly, phenotyping of the viruses in low CD4-expressing
primary microglia revealed that most CSF and plasma
clones were unable to efficiently enter these cells, although
CSF-derived viral clones were overall better in entering
microglial cells than the plasma-derived clones [Fig.3].
The enhanced ability of CSF clones, to infect microglia was
more pronounced in compartmentalized subject 19 [Fig.3b].
While both BaL and YU-2 are R5 M-tropic viruses, BaL
was isolated from infant lung tissue (Gartner etal. 1986),
whereas YU-2 was isolated from brain tissue (Li etal. 1991)
and hereby potentially more representative of the neuro-
tropic viruses in the CNS. From this perspective, we com-
pared the infection of the viral clones to YU-2 and found
that each subject had one CSF clone with an intermediate
M-tropic phenotype, defined as ≥ 50% of YU-2 infection, for
cell entry in microglia [Fig.4]. In subject 27, we also found
one plasma clone with this intermediate M-tropic entry phe-
notype. The plasma clone had a higher FPR than the CSF
clone with a similar intermediate phenotype (71.1 vs. 58.6),
suggesting two separate virus populations possibly originat-
ing from different low CD4- expressing cells.
Discussion
With up to 43% of the HIV-infected population still
affected by lasting HIV-associated neurological impair-
ments despite viral suppression with ART, research on the
neuropathogenesis of HIV remains essential (Wang etal.
2020). In this study, we report, for the first time, that patient-
derived replication-competent HIV variants can infect and
replicate in human primary microglia, however, HIV replica-
tion was more efficient in primary CD4+ T-cells.
HIV RNA can be detected in the CSF as early as 8days
post-estimated infection, however, RNA levels in CSF are
generally lower than in plasma (Valcour etal. 2012). Within
our study population, HIV RNA levels were significantly
lower in CSF than in plasma, however, 16% (n = 3) of the
subjects (subjects 12, 17, 19) had higher virus concentra-
tion in CSF than in plasma, a phenomenon associated with
HAND (Bai etal. 2017). Among these three subjects, only
subject 17 had neurological symptoms at the time of sam-
pling (HIV encephalopathy). A recent multicenter study
reported that up to 30% of treatment-naïve individuals with
HIV-associated dementia (HAD) had CSF to plasma HIV
RNA discordance (Ulfhammer etal. 2022). The detection
of higher levels of HIV RNA in CSF than in plasma sug-
gests compartmentalized viral production and/or replication
in CNS resident cells.
Compartmentalization is observed in some but not all
subjects. We found a genetically compartmentalized viral
population in the CSF in 2 subjects (subjects 13 and 19). It
is thought that within the first two years of infection, CSF
compartmentalized variants are predominantly R5 T-tropic
and associated with clonal amplification and the presence
of elevated CSF pleocytosis (Sturdevant etal. 2015). HIV
infection may progress in advanced stages of disease to
HAD, in which both compartmentalized R5 T-tropic and
R5 M-tropic CSF viral populations can be detected in the
CSF (Schnell etal. 2011). Based on genetic analysis, these
R5 M-tropic viruses are more genetically diverse than the
R5 T-tropic viruses, which suggests that they are replicat-
ing in the long-lived cells of the CNS (Arrildt etal. 2015;
Schnell etal. 2011). In our study, subjects 13 and 19 both had
compartmentalized R5-using CSF viruses, however, only
subject 13 had reported neurological symptoms at the time
of sampling, suggesting distinct viral tropism between the
subjects, namely R5 M-tropic (subject 13) and R5 T-tropic
(subject 19). In addition, an X4-using viral population was
found in the CSF of subject 17 who was diagnosed with HIV
encephalopathy. As the only subject with severe neurological
symptoms, it remains to be determined whether the preva-
lence of X4-using virus in the CNS is associated with the
progression of neurological disease.
In the CNS, HIV is primarily detected in perivascular
macrophages and primary microglia that both express the
CCR5 co-receptor (Joseph etal. 2015). In this study, we
phenotypically characterized CSF- and plasma-derived viral
clones from compartmentalized subjects 19 and equilibrated
subjects 8 and 27 for their ability to infect and replicate in
CD4+ T-cells and primary microglia. Characteristic of both
Journal of NeuroVirology
Donor 1Donor 2Donor 3
BaL
YU-2
JRCSF
1
3
4
6
1
3
5
7
10
100
1000
10000
100000
1000000
CSFViral clones Plasma
28.8 44.2 28.8 28.8 7.8 7.8 7.8 7.8
BaL
YU-2
JRCSF
6
8
1
2
3
4
7
10
100
1000
10000
100000
1000000
CSFViral clones Plasma
68.8 68.8 0.4 0.4 56.9 0.4 56.9
BaL
YU-2
JRCSF
1
3
4
2
8
23
10
100
1000
10000
100000
1000000
CSFViral clones Plasma
61.5 58.6 43.3 71.1 71.1 43.3
BaL
YU-2
JRCSF
1
3
4
6
1
3
5
7
0
50
100
150
200
Viral clones
CSF Plasma
no ART+ MVC
BaL
YU-2
JRCSF
6
8
1
2
3
4
7
0
50
100
150
200
Viral clones
CSF Plasma
BaL
YU-2
JRCSF
1
3
4
2
8
23
0
50
100
150
200
Viral clones
CSF Plasma
FPR
FPR
FPR
Subject 8
Luminescence
Subject 19
Luminescence
Subject27
Luminescence
%Infection %Infection %Infection
ad
b
c
e
f
Fig. 2 CSF- and plasma-derived viruses can efficiently infect and rep-
licate in CD4+ T-cells. a-c CD4+ T-cells were infected with 10 ng
(p24 Gag) CSF- or plasma-derived virus generated from subjects
8, 19 and 27. Scattered dot plots depict luciferase activity measured
in supernatant collected on day 14 post-infection, whereas horizon-
tal black lines represent median luminescence. FPR values for each
clone are included at the bottom of the figure. An FPR value below
3.5% is predicted to be X4-tropic virus. An FPR value above 3.5%
is predicted to be R5-tropic virus. (d-f) CD4+ T-cells were untreated
(no ART) or treated with 100nM Maraviroc (MVC) prior to infec-
tion with 10 ng (p24 Gag) CSF- or plasma-derived virus generated
from subjects 8, 19 and 27. Bar graphs depict the maximum infection
measured on day 14 post-infection with (grey) and without (white)
treatment with MVC. Black error bars depict standard error of the
means. Lab strains and viruses derived from plasma (red) or CSF
(blue) are separated by vertical dotted lines
Journal of NeuroVirology
Fig. 3 CSF-derived viruses
have a modest enhancement for
infecting primary microglia. a-c
Primary microglia were infected
with 10ng (p24 Gag) virus gen-
erated from subject 8, 19 and
27. Graphs represent luciferase
activity measured in supernatant
collected on Day 17 post-
infection. Black line depicts the
median luminescence measured.
Lab strains and viruses derived
from plasma (red) or CSF (blue)
are separated by vertical dotted
lines
Bal
YU-2
JRCSF
1
3
4
6
1
3
5
7
1
10
100
1000
10000
100000
1000000
Subject 8
Luminescence
Viral clones
CSFPlasma
FPR
28.8 44.2 28.8 28.8 7.8 7.8 7.8 7.8
Bal
YU-2
JRCSF
6
8
1
2
3
4
7
1
10
100
1000
10000
100000
1000000
Subject 19
Luminescence
Viral clonesCSFPlasma
68.8 68.8 0.4 0.4 56.9 0.4 56.9 FPR
Bal
YU-2
JRCSF
1
3
4
2
8
2.3
1
10
100
1000
10000
100000
1000000
Subject 27
Luminescence
Viral clonesCSFPlasma
61.5 58.6 43.3 71.1 71.1 43.3FPR
Donor 1
Donor 2
Donor 1
Donor 2
Donor
1
Donor 2
Journal of NeuroVirology
M- and T-tropic viruses, all viral clones were able to effec-
tively infect high CD4-expressing T-cells with no major dif-
ferences between the CSF and the plasma viruses. Treatment
with MVC confirmed productive infection and corroborated
the prediction of both X4 and R5-using viruses in the plasma
of subject 19 and revealed a possible dual-tropic viral
population in subject 8. Interestingly, we also observed an
enhanced infection of the X4-using viruses following treat-
ment with MVC, suggesting that treatment of CD4+ T-cells
with MVC increases their susceptibility to X4-using viral
infection possibly due to cell activation (López-Huertas
etal. 2020; Madrid-Elena etal. 2018).
Microglia CD4
1
10
100
1000
10000
100000
1000000
Labstrains
D14 p.i.
Luminescence
BaL
YU-2
JRCSF
Microglia CD4
1
10
100
1000
10000
100000
1000000
Subject 8
D14 p.i.
Luminescence
1
3
4
6
1
3
5
7
Microglia CD4
1
10
100
1000
10000
100000
Subject 19
D14 p.i.
Luminescence
6
8
1
2
3
4
7
Microglia CD4
1
10
100
1000
10000
100000
1
3
4
2
8
2.3
Subject 27
D14 p.i.
Luminescence
Fig. 4 Majority of the CSF and plasma viral population displayed the
typical R5 T-cell tropic phenotype. Graphs represent luciferase activ-
ity measured in supernatant collected on Day 14 post-infection (D14
p.i.) in primary microglia and CD4+ T-cells. Laboratory strains are
depicted in black, CSF-derived clones in blue and plasma-derived
clones in red. Black arrows indicate viral clones with an intermediate
M-tropic phenotype
Journal of NeuroVirology
Furthermore, In line with previous studies on monocyte-
derived macrophages and Affinofile cells (Brese etal. 2018;
Gonzalez-Perez etal. 2012; Schnell etal. 2011; Sturdevant
etal. 2012), CSF-derived viral clones were overall more
efficient at infecting low CD4-expressing primary microglia
than the plasma-derived clones, despite differences among
donors. The infection levels of the CSF-derived clones,
however, never reached the level of the R5 M-tropic labora-
tory strains Bal and YU-2, and therefore did not meet the
criteria for an M-tropic phenotype. R5 T-cell tropic viruses
found in the CSF are presumed to originate from infiltrat-
ing infected CD4+ T-cells or are potentially produced by
resident CD4+ T-cells in the brain parenchyma (Joseph and
Swanstrom 2018; Schnell etal. 2011). Nonetheless, we
observed an intermediate M-tropic phenotype, defined
as ≥ 50% of YU-2 infection, for several CSF clones
(one in each subject) and one plasma clone. Other than
CSF and plasma (Arrildt etal. 2015; Joseph etal. 2014;
Sturdevant etal. 2015), viruses with an intermediate
M-tropic phenotype, determined by low CD4 Affinofile
cells and/or monocyte-derived macrophages, have also
been detected in peripheral tissues, such as the colon, lungs,
and lymph nodes (Brese etal. 2018). Due to the relatively
invasive nature of CSF collection, longitudinal samples
were not obtained, therefore we were not able to determine
whether this intermediate M-tropic phenotype represents
an evolutionary intermediate on the path to macrophage
tropism. A recent paper by Woodburn etal. reported that
patient-derived M-tropic HIV Env proteins confer an entry
advantage over T cell-tropic Envs when infecting primary
microglia (Woodburn etal. 2022). In line with our study,
infection of primary microglia with an R5 T-cell tropic virus
with an intermediate M-tropic phenotype approached but did
not reach the infection level of the M-tropic viruses.
It is hypothesized that the enhanced ability of M-tropic
viruses to utilize low CD4 surface expression for viral
entry is marked by an increased Env: CD4 affinity,
enhanced sensitivity to sCD4 inhibition, and other subtle
changes in the trimer conformation of the Env protein.
Several studies have reported a variety of substitutions in
the envelope gene found to be associated with M-tropic
CNS-derived viruses, such as N283 in the CD4 binding
site (CD4bs) (Dunfee etal. 2006), a conserved amino
acid in the V1 loop (Musich etal. 2011), and the loss
of an N-linked glycosylation site at 386 (Duenas-Decamp
etal. 2009; Dunfee etal. 2007). However, none of these
genetic mutations were conserved across different studies.
Interestingly, non-M-tropic viruses were recently shown
to productively and efficiently infect macrophages through
Env-dependent cell–cell fusion with infected CD4+ T-cells
(Han etal. 2022). The Envs expressed on infected T-cells
also showed enhanced interaction with the CD4 and CCR5
receptors and were less dependent on the surface density,
compared to the cell-free virus-associated Envs. However,
the infection of microglial cells invivo through cell-to-cell
fusion with infiltrating infected CD4+ T-cells is yet to be
demonstrated. In addition, the limited infection of primary
microglia observed with both M- and T-tropic viruses can
also be attributed to the host-restriction factors expressed
in microglia, such as Sp3 protein and C-EBPγ, that func-
tion as transcriptional repressors (Wallet etal. 2019).
Furthermore, we recognize that our study has several
limitations. First, this study, utilized plasma and CSF
samples obtained for clinical diagnosis, which limited the
number of participants that could be included. Second, in
this small study sample, we were not able to establish a
relation between clinical symptoms and CNS diversifica-
tion. Third, we used the Env protein to generate recombi-
nant viral clones rather than using full-length viral clones.
While the envelope protein is the major determinant for
co-receptor usage and CD4 binding, we cannot completely
rule out the possibility of viral evolution outside of the Env
gene that contributes to the M-tropic phenotype. Finally,
we used a cell-free virus infection assay which might not
fully represent the modes of microglia infection invivo.
Nonetheless, we were able to derive significant and
compelling evidence that supports the CNS as a viral res-
ervoir for HIV in a subset of patients. Among these find-
ings is the detection of a genetically distinct CSF viral
population, indicating viral replication in the CNS. In
addition, we detected CSF-derived viral clones that exhibit
a modestly enhanced ability to enter primary microglia.
Ultimately, the evidence of viral replication and evolu-
tion in the CNS highlights the importance of the CNS as
a HIV reservoir.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s13365- 024- 01207-w .
Acknowledgements The authors thank the team at the Netherlands
Brain Bank for their services. We thank the study participants for their
participation in the research study.
Funding This research was funded by Aidsfonds grant numbers
P-13204 and P-37203, Health Holland grant numbers LSHM2OO12-
SGF and LSHM19101-SGF.
Data availability The data that support the findings of this study are
available from the corresponding author upon reasonable request.
Declarations
Institutional review board statement Fresh postmortem adult human
brain tissue was provided by the Netherlands Brain Bank (NBB). All
subjects gave their informed consent for inclusion before they partici-
pated in the study. The study was conducted in accordance with the
Declaration of Helsinki, and the protocol was approved by the Ethics
Committee of VU Medical Center (VUMC, Amsterdam, The Nether-
lands). Paired CSF and plasma samples were obtained as part of the
AIDS Therapy Evaluation in the Netherlands (ATHENA) cohort which
includes patients by an opt-out principle.
Journal of NeuroVirology
Informed consent statement Informed consent was obtained from all
subjects involved in the study.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
References
Arrildt KT, LaBranche CC, Joseph SB, Dukhovlinova EN, Graham
WD, Ping LH, Schnell G, Sturdevant CB, Kincer LP, Mallewa
M, Heyderman RS, Van Rie A, Cohen MS, Spudich S, Price RW,
Montefiori DC, Swanstrom R (2015) Phenotypic correlates of
HIV-1 macrophage tropism. J Virol 89:11294–11311. https:// doi.
org/ 10. 1128/ jvi. 00946- 15
Bai F, Iannuzzi F, Merlini E, Borghi L, Tincati C, Trunfio M, Bini T,
d’Arminio Monforte A, Marchetti G (2017) Clinical and viro-
immunological correlates of HIV associated neurocognitive dis-
orders (HAND) in a cohort of antiretroviral-naïve HIV-infected
patients. AIDS 31:311–314. https:// doi. org/ 10. 1097/ QAD.
00000 00000 001346
Bednar MM, Sturdevant CB, Tompkins LA, Arrildt KT, Dukhovlinova
E, Kincer LP, Swanstrom R (2015) Compartmentalization, viral
evolution, and viral latency of HIV in the CNS. Curr HIV/AIDS
Rep 12:262–271. https:// doi. org/ 10. 1007/ s11904- 015- 0265-9
Boom R, Sol JA, Salimans MMM, Jansen CL, Wertheim-Van Dillen
PME, Van Der Noordaa J (1990) Rapid and simple method for
purification of nucleic acids. J Clin Microbiol 28:495–503
Borrajo A, Svicher V, Salpini R, Pellegrino M, Aquaro S (2021) Cru-
cial role of central nervous system as a viral anatomical compart-
ment for hiv-1 infection. Microorganisms 9:2537. https:// doi. org/
10. 3390/ micro organ isms9 122537
Brese RL, Gonzalez-Perez MP, Koch M, O’Connell O, Luzuriaga K,
Somasundaran M, Clapham PR, Dollar JJ, Nolan DJ, Rose R,
Lamers SL (2018) Ultradeep single-molecule real-time sequenc-
ing of HIV envelope reveals complete compartmentalization
of highly macrophage-tropic R5 proviral variants in brain and
CXCR4-using variants in immune and peripheral tissues. J Neu-
rovirol 24:439–453. https:// doi. org/ 10. 1007/ s13365- 018- 0633-5
Chan P, Spudich S (2022) HIV compartmentalization in the CNS and its
impact in treatment outcomes and cure strategies. Curr HIV/AIDS
Rep 19:207–216. https:// doi. org/ 10. 1007/ s11904- 022- 00605-1
Clifford DB, Ances BM (2013) HIV-associated neurocognitive disor-
der. Lancet Infect Dis 13:976–986
Duenas-Decamp MJ, Peters PJ, Burton D, Clapham PR (2009) Deter-
minants flanking the CD4 binding Loop modulate macrophage
tropism of human immunodeficiency virus type 1 R5 envelopes.
J Virol 83:2575–2583. https:// doi. org/ 10. 1128/ jvi. 02133- 08
Dunfee RL, Thomas ER, Gorry PR, Wang J, Taylor J, Kunstman K,
Wolinsky SM, Gabuzda D (2006) The HIV Env variant N283
enhances macrophage tropism and is associated with brain infec-
tion and dementia. PNAS 103:15160–15165
Dunfee RL, Thomas ER, Wang J, Kunstman K, Wolinsky SM, Gabuzda
D (2007) Loss of the N-linked glycosylation site at position 386 in
the HIV envelope V4 region enhances macrophage tropism and is
associated with dementia. Virology 367:222–234. https:// doi. org/
10. 1016/j. virol. 2007. 05. 029
Gartner S, Markovits P, Markovitz DM, Kaplan MH (1986) The role
of mononuclear phagocytes in HTLV-III/LAV infection. Science
233:215–219. https:// doi. org/ 10. 1126/ scien ce. 30146 48
Gonzalez-Perez MP, O’Connell O, Lin R, Sullivan WM, Bell J,
Simmonds P, Clapham PR (2012) Independent evolution of
macrophage-tropism and increased charge between HIV-1 R5
envelopes present in brain and immune tissue. Retrovirology 9:20.
https:// doi. org/ 10. 1186/ 1742- 4690-9- 20
Gumbs SBH, Kübler R, Gharu L, Schipper PJ, Borst AL, Snijders
GJLJ, Ormel PR, van Berlekom AB, Wensing AMJ, de Witte
LD, Nijhuis M (2022) Human microglial models to study HIV
infection and neuropathogenesis: a literature overview and com-
parative analyses. J Neurovirol 28:64–91. https:// doi. org/ 10. 1007/
s13365- 021- 01049-w
Han M, Cantaloube-Ferrieu V, Xie M, Armani-Tourret M, Woottum M,
Pagès JC, Colin P, Lagane B, Benichou S (2022) HIV-1 cell-to-
cell spread overcomes the virus entry block of non-macrophage-
tropic strains in macrophages. PLoS Pathog 18(5):e1010335.
https:// doi. org/ 10. 1371/ journ al. ppat. 10103 35
Heaton RK, Clifford DB, Franklin DR, Woods BSP, Ake PC, Vaida F,
Ellis RJ, Letendre SL, Marcotte TD, Atkinson JH, Rivera-Mindt
M, Vigil OR, Taylor MJ, Collier AC, Marra CM, Gelman BB,
Mcarthur JC, Morgello MS, Simpson DM, Grant I (2010) HIV-
associated neurocognitive disorders persist in the era of potent
antiretroviral therapy CHARTER Study. Neurology 75:2087–2096
Hudson RR (2000) A New Statistic for detecting genetic differentiation.
Genetics 155:2011–2014
Hudson RR, Slatkint M, Maddison WP (1992) Estimation of levels
of Gene Flow from DNA sequence data. Genetics 132:583–589
Jadhav S, Nema V (2021) HIV-Associated neurotoxicity: the interplay
of host and viral proteins. Mediators Inflamm. https:// doi. org/ 10.
1155/ 2021/ 12670 41
Joseph SB, Swanstrom R (2018) The evolution of HIV-1 entry pheno-
types as a guide to changing target cells. J Leukoc Biol 103:421–
431. https:// doi. org/ 10. 1002/ JLB. 2RI05 17- 200R
Joseph SB, Arrildt KT, Swanstrom AE, Schnell G, Lee B, Hoxie JA,
Swanstrom R (2014) Quantification of Entry phenotypes of mac-
rophage-tropic HIV-1 across a wide range of CD4 densities. J
Virol 88:1858–1869. https:// doi. org/ 10. 1128/ jvi. 02477- 13
Joseph SB, Arrildt KT, Sturdevant CB, Swanstrom R (2015) HIV-1
target cells in the CNS. J Neurovirol 21:276–289. https:// doi. org/
10. 1007/ s13365- 014- 0287-x
Katoh K, Rozewicki J, Yamada KD (2018) MAFFT online service:
multiple sequence alignment, interactive sequence choice and
visualization. Brief Bioinform 20:1160–1166. https:// doi. org/ 10.
1093/ bib/ bbx108
Ko A, Kang G, Hattler JB, Galadima HI, Zhang J, Li Q, Kim WK
(2019) Macrophages but not astrocytes Harbor HIV DNA in the
brains of HIV-1-Infected aviremic individuals on suppressive
antiretroviral therapy. J Neuroimmune Pharmacol 14:110–119.
https:// doi. org/ 10. 1007/ s11481- 018- 9809-2
Kosakovsky Pond SL, Frost SDW, Muse SV (2005) HyPhy: hypothesis
testing using phylogenies. Bioinformatics 21:676–679. https:// doi.
org/ 10. 1093/ bioin forma tics/ bti079
Lamers SL, Rose R, Maidji E, Agsalda-Garcia M, Nolan DJ, Fogel
GB, Salemi M, Garcia DL, Bracci P, Yong W, Commins D, Said
J, Khanlou N, Hinkin CH, Sueiras MV, Mathisen G, Donovan S,
Shiramizu B, Stoddart CA, Singer EJ (2016) HIV DNA is fre-
quently present within Pathologic Tissues Evaluated at autopsy
from combined antiretroviral therapy-treated patients with unde-
tectable viral loads. J Virol 90:8968–8983. https:// doi. org/ 10.
1128/ jvi. 00674- 16
Journal of NeuroVirology
Li Y, Kappes JC, Conway JA, Price RW, Shaw GM, Hahn BH (1991)
Molecular characterization of human immunodeficiency virus
type 1 cloned directly from uncultured human brain tissue: iden-
tification of replication-competent and-defective viral genomes.
J Virol 65:3973–3985
López-Huertas MR, Jiménez-Tormo L, Madrid-Elena N, Gutiérrez C,
Vivancos MJ, Luna L, Moreno S (2020) Maraviroc reactivates
HIV with potency similar to that of other latency reversing drugs
without inducing toxicity in CD8 T cells. Biochem Pharmacol
182:114231. https:// doi. org/ 10. 1016/j. bcp. 2020. 114231
Madrid-Elena N, Laura García-Bermejo M, Serrano-Villar S, Díaz-
De Santiago A, Sastre B, Gutiérrez C, Dronda F, Díaz MC,
Domínguez E, Rosa López-Huertas M, Hernández-Novoa B,
Moreno S (2018) Maraviroc is Associated with latent HIV-1
reactivation through NF-B activation in resting CD4 T cells from
HIV-Infected individuals on suppressive antiretroviral therapy. J
Virol 92:e01931–e01917. https:// doi. org/ 10. 1128/ JVI. 01931- 17
Mattei D, Ivanov A, van Oostrum M, Pantelyushin S, Richetto J,
Mueller F, Beffinger M, Schellhammer L, vom Berg J, Wollscheid
B, Beule D, Paolicelli RC, Meyer U (2020) Enzymatic
dissociation induces transcriptional and proteotype bias in brain
cell populations. Int J Mol Sci 21:1–20. https:// doi. org/ 10. 3390/
ijms2 12179 44
Musich T, Peters PJ, Duenas-Decamp MJ, Gonzalez-Perez MP, Robinson
J, Zolla-Pazner S, Ball JK, Luzuriaga K, Clapham PR (2011) A
conserved determinant in the V1 Loop of HIV-1 modulates the
V3 Loop to Prime low CD4 use and macrophage infection. J Virol
85:2397–2405. https:// doi. org/ 10. 1128/ jvi. 02187- 10
Schnell G, Joseph S, Spudich S, Price RW, Swanstrom R (2011) HIV-1
replication in the central nervous system occurs in two distinct cell
types. PLoS Pathog 7:e1002286. https:// doi. org/ 10. 1371/ journ al.
ppat. 10022 86
Slatkin M, Maddison WP (1989) A cladistic measure of Gene Flow
inferred from the phylogenies of alleles. Genetics 123:603–613
Sturdevant CB, Dow A, Jabara CB, Joseph SB, Schnell G, Takamune
N, Mallewa M, Heyderman RS, Van Rie A, Swanstrom R (2012)
Central Nervous System compartmentalization of HIV-1 subtype
C variants early and late in infection in Young Children. PLoS
Pathog 8:e1003094. https:// doi. org/ 10. 1371/ journ al. ppat. 10030 94
Sturdevant CB, Joseph SB, Schnell G, Price RW, Swanstrom R,
Spudich S (2015) Compartmentalized replication of R5 T cell-
tropic HIV-1 in the Central Nervous System early in the course
of infection. PLoS Pathog 11:1–24. https:// doi. org/ 10. 1371/
journ al. ppat. 10047 20
Swenson LC, Mo T, Dong WWY, Zhong X, Woods CK, Jensen MA,
Thielen A, Chapman D, Lewis M, James I, Heera J, Valdez H,
Harrigan PR (2011) Deep sequencing to infer HIV-1 co-receptor
usage: application to three clinical trials of maraviroc in treat-
ment-experienced patients. J Infect Dis 203:237–245. https:// doi.
org/ 10. 1093/ infdis/ jiq030
Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolu-
tionary Genetics Analysis Version 11. Mol Biol Evol 38:3022–
3027. https:// doi. org/ 10. 1093/ molbev/ msab1 20
Tso FY, Kang G, Kwon EH, Julius P, Li Q, West JT, Wood C (2018)
Brain is a potential sanctuary for subtype C HIV-1 irrespective of
ART treatment outcome. PLoS ONE 13:e0201325. https:// doi.
org/ 10. 1371/ journ al. pone. 02013 25
Ulfhammer G, Edén A, Antinori A, Brew BJ, Calcagno A, Cinque
P, De Zan V, Hagberg L, Lin A, Nilsson S, Oprea C, Pinnetti C,
Spudich S, Trunfio M, Winston A, Price RW, Gisslén M (2022)
Cerebrospinal fluid viral load across the spectrum of untreated
human immunodeficiency virus type 1 (HIV-1) infection: a cross-
sectional Multicenter Study. Clin Infect Dis 75:493–502. https://
doi. org/ 10. 1093/ cid/ ciab9 43
Valcour V, Chalermchai T, Sailasuta N, Marovich M, Lerdlum S,
Suttichom D, Suwanwela NC, Jagodzinski L, Michael N, Spudich
S, Van Griensven F, De Souza M, Kim J, Ananworanich J (2012)
Central nervous system viral invasion and inflammation during
acute HIV infection. J Infect Dis 206:275–282. https:// doi. org/
10. 1093/ infdis/ jis326
Wallet C, De Rovere M, Van Assche J, Daouad F, De Wit S, Gautier
V, Mallon PWG, Marcello A, Van Lint C, Rohr O, Schwartz C
(2019) Microglial cells: the Main HIV-1 Reservoir in the brain.
Front Cell Infect Microbiol 9:362. https:// doi. org/ 10. 3389/ fcimb.
2019. 00362
Wang Y, Liu M, Lu Q, Farrell M, Lappin JM, Shi J, Lu L, Bao Y (2020)
Global prevalence and burden of HIV-associated neurocognitive
disorder: a meta-analysis. Neurology 95:E2610–E2621. https://
doi. org/ 10. 1212/ WNL. 00000 00000 010752
Woodburn BM, Kanchi K, Zhou S, Colaianni N, Joseph SB, Swanstrom
R (2022) Characterization of macrophage-tropic HIV-1 infection
of Central Nervous System cells and the influence of inflamma-
tion. J Virol. https:// doi. org/ 10. 1128/ jvi. 00957- 22
Zárate S, Pond SLK, Shapshak P, Frost SDW (2007) Comparative
study of methods for detecting sequence compartmentalization
in human immunodeficiency virus type 1. J Virol 81:6643–6651.
https:// doi. org/ 10. 1128/ jvi. 02268- 06
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