| Editor’s Pick | Virology | Research Article

APOBEC3 degradation is the primary function of HIV-1 Vif
determining virion infectivity in the myeloid cell line THP-1
Terumasa Ikeda,1 Ryo Shimizu,1,2 Hesham Nasser,1,3 Michael A. Carpenter,4,5 Adam Z. Cheng,6,7 William L. Brown,6,7 Daniel Sauter,8Reuben S. Harris4,5
AUTHOR AFFILIATIONS See affiliation list on p. 18.

IMPORTANCE HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However,
it is currently unclear whether Vif has additional essential cellular targets. To address thisquestion, we disrupted A3A to A3G genes in the THP-1 myeloid cell line using CRISPR
and compared the infectivity of wild-type HIV-1 and Vif mutants with the selective A3
neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1
and the other Vif mutants is fully restored by ablating the expression of cellular A3A to
A3G proteins. These results indicate that A3 proteins are the only essential target of Vif
that is required for fully infectious HIV-1 production from THP-1 cells.

KEYWORDS HIV-1, APOBEC3, G-to-A mutations, deaminase-dependent mechanism,
deaminase-independent mechanism, VifT

he A3 family of proteins comprise seven single-strand DNA cytosine deaminases(A3A–A3D and A3F–A3H) in humans (1–3). A3 enzymes have broad and essential
roles in innate antiviral immunity against parasitic DNA-based elements (4–6). Retrovi­
ruses are sensitive to A3 enzyme activity due to the obligate step of reverse transcription
during viral replication that produces single-stranded cDNA intermediates. These viral
cDNA intermediates can act as substrates for A3 enzymes, as demonstrated by C-to-U
deamination resulting in G-to-A mutations in the genomic strand. To date, the best-char­acterized substrate of A3 enzymes is human immunodeficiency virus type 1 (HIV-1). In
CD4+ T lymphocytes, four A3 proteins (A3D, A3F, A3G, and stable A3H haplotypes) restrict
HIV-1 replication by mutating viral cDNA intermediates and by physically blocking
reverse transcription (7–14). A3 enzymes have a preference for specific dinucleotide
Month XXXX Volume 0

Issue 0Invited Editor Stuart J. Neil, King's College London,
London, United Kingdom
Editor Stephen P. Goff, Columbia University Medical
Center, New York, New York, USA
Address correspondence to Terumasa Ikeda,
ikedat@kumamoto-u.ac.jp.
Terumasa Ikeda and Ryo Shimizu contributed equallyto this article. Author order was determined in orderof increasing seniority.
The authors declare no conflict of interest.
See the funding table on p. 18.
Received 28 March 2023
Accepted 22 June 2023
Published 9 August 2023
Copyright © 2023 Ikeda et al. This is an open-accessarticle distributed under the terms of the Creative
Commons Attribution 4.0 International license.
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ABSTRACT HIV-1 must overcome multiple innate antiviral mechanisms to replicate
in CD4+ T lymphocytes and macrophages. Previous studies have demonstrated thatthe apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) familyof proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1
restriction in CD4+ T lymphocytes. Virus-encoded virion infectivity factor (Vif ) coun­teracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in
infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4+
T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades
only A3 proteins during viral replication is currently unknown. Herein, we describe thedevelopment and characterization of A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells.
In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced
infectivity in parental and A3F-null THP-1 cells, and a more modest decrease in infectivity
in A3F/A3G-null cells. Remarkably, disruption of A3A–A3G protein expression completely
restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate thatthe primary function of Vif during infectious HIV-1 production from THP-1 cells is thetargeting and degradation of A3 enzymes.

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motifs (5´-CC for A3G and 5´-TC for other A3 enzymes) at target cytosine bases, whichappear as 5´-AG or 5´-AA mutations in the genomic strand (7, 8, 15, 16).
Virus-encoded virion infectivity factor (Vif ) functions in disrupting the activity of A3
enzymes. Vif forms an E3 ubiquitin ligase complex that degrades A3 enzymes througha proteasome-mediated pathway (2, 3, 17, 18). The central domain of this complex isa Vif heterodimer with the transcription factor, core binding factor subunit β (CBF-β),which stabilizes Vif during disruption of A3 protein activity (19, 20). Vif also suppresses
the transcription of A3 enzymes by hijacking RUNX/CBF-β complex (21). In addition tothese Vif-dependent mechanisms, HIV-1 reverse transcriptase and protease have beenshown to disrupt the activity of A3 enzymes via Vif-independent mechanisms (22, 23).
Recently, functional proteomic analyses have demonstrated that Vif has several target
proteins, including the PPP2R5 family of proteins, in CD4+ T cell lines and lymphocytes(24, 25). These findings indicate that Vif may have additional essential target proteinsduring HIV-1 infection.
We previously reported that endogenous A3G protein contributes to HIV-1 restric­tion in a deaminase-dependent manner in THP-1 cells (26). Although disruption of theA3G gene nearly eliminates viral G-to-A mutations, Vif-deficient HIV-1 virions have 50%
lower infectivity than wild-type HIV-1 or mutants selectively lacking A3G degradation
activity (26). These results indicated that Vif-mediated inhibition of A3G and at least one
additional A3 proteins is required for efficient infectious HIV-1 production.
In the present study, we evaluate the effects of other A3 proteins on HIV-1 infectiv­
ity by developing and characterizing A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells
using HIV-1 Vif mutants with selective A3 neutralization activities. In comparison towild-type HIV-1, Vif-deficient HIV-1 infectivity is strongly inhibited in A3F-null THP-1 cells
and modestly inhibited in A3F/A3G-null THP-1 cells. In contrast, an HIV-1 Vif mutant
selectively lacking A3F degradation activity had comparable infectivity to wild-type
HIV-1 in A3F-null THP-1 cells and 50% infectivity in parental THP-1 cells, indicating thatA3F protein contributes to HIV-1 restriction in THP-1 cells. Furthermore, Vif-deficientHIV-1 infectivity is comparable to wild-type HIV-1 in A3A-to-A3G-null THP-1 cells. These
results demonstrate that A3 proteins are the primary target of HIV-1 Vif during infectious
virus production from THP-1 cells.

Endogenous A3H protein is not involved in HIV-1 restriction in THP-1 cells
THP-1 cells express significant levels of A3B, A3C, A3F, A3G, and A3H mRNA (26). The
results of our previous study indicated that A3G and at least one additional A3 proteins
are involved in HIV-1 restriction in THP-1 cells (26). Variations in the amino acid sequenceof A3 family proteins are known to influence HIV-1 restriction activity (27), and theA3H gene is the most polymorphic of all human A3 genes (10, 22, 28, 29). The A3H
allele is grouped into stable and unstable haplotypes according to the combinationof amino acid residues at positions 15, 18, 105, 121, and 178 (10, 22, 28, 29). Stable
A3H haplotypes are active against HIV-1, whereas unstable A3H haplotypes have absent
or minimal activity as they encode proteins with low stability (9, 10, 22, 29, 30). To
determine A3H genotypes, we sequenced A3H cDNA from THP-1 cells. Sequencing data
identified an unstable haplotype in the THP-1 genome, termed A3H hapI gene (Fig. 1A).
These data suggest that endogenous A3H protein has minimal restriction activity against
Vif-deficient HIV-1 in THP-1 cells.
The A3H hapI protein results in expression of an unstable protein that has weak antiHIV-1 activity (28, 29, 31). However, this protein is enzymatically active and has an HIV-1
restriction phenotype similar to the stable A3H haplotype, A3H hapII protein, when bothproteins are expressed at the same levels (31). In addition, A3H protein expression levels
are upregulated during HIV-1 infection (10, 22), and A3H hapI protein is resistant to Vifmediated degradation (32). Accordingly, we evaluated whether the expression of A3H
hapI protein is associated with HIV-1 restriction in THP-1 cells. To address this question,
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FIG 1 Endogenous A3H protein does not inhibit HIV-1 in THP-1 cells. (A) A3H haplotypes in THP-1 cells. The indicated positions are key amino acid residues
that determine the expression of unstable (hapI) or stable (hapII) A3H protein. (B) Schematic of the susceptibility of HIV-1 Vif mutants to antiviral activity bystable A3H haplotypes. Key amino acid residues of Vif that determine the susceptibility of HIV-1 IIIB to restriction by stable A3H haplotypes. −, full resistance; +,partial resistance; +++, sensitivity. (C) Schematic depiction of the pseudo-single cycle infectivity assay. For details, see the main text and the “Pseudo-single cycle
infectivity assays” section in Materials and Methods. (D) Representative infectivity of HIV-1 mutants with hyper- and hypo-functional Vif produced from THP-1
cells. Top panels show the infectivity of hyper-Vif, hypo-Vif, IIIB Vif, and Vif-deficient HIV-1 mutants produced in THP-1 cells. The amounts of produced viruses
used to infect TZM-bl cells were normalized to p24 levels. Each bar shows the average of four independent experiments with standard deviation (SD). Data arerepresented as relative infectivity compared to hyper-Vif HIV-1. Statistical significance was determined using the two-sided paired t test. *P < 0.05 comparedwith the infectivity of hyper-Vif HIV-1. The bottom panels are representative western blots of three independent experiments. The levels of viral and cellular
proteins in viral particles and whole-cell lysates are shown. p24 and HSP90 were used as loading controls. (E) G-to-A mutations. Average number of G-to-A
mutations in the 564 bp pol gene after infection with hyper-Vif, hypo-Vif, IIIB Vif, or Vif-deficient HIV-1 produced from THP-1. Each bar depicts the average of three
independent experiments with SD. (F) G-to-A mutation profile. Dinucleotide sequence contexts of G-to-A mutations in the 564 bp pol gene after infection with
the indicated viruses produced from indicated cell lines. Each vertical line indicates the location of the dinucleotide sequence contexts described in the legend
within the 564 bp amplicon (horizontal line).

we utilized HIV-1 Vif mutants that selectively degrade stable A3H protein (hyperfunctional Vif; hyper-Vif ) or lack stable A3H degradation (hypo-functional Vif; hypo-Vif )(10) (Fig. 1B). IIIB Vif displays an intermediate phenotype (Fig. 1B). Of note, hyper-Vif,hypo-Vif, and IIIB Vif have full neutralization activity against A3D, A3F, and A3G proteins(10).
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To allow efficient and equivalent delivery of HIV-1 to THP-1 and its derivatives,
vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped full-length HIV-1 andits Vif mutants were produced from 293T cells and titrated with CEM-GXR cells fordetermination of the multiplicity of infection (MOI) as described (23, 26) (Fig. 1C). Then,these VSV-G pseudotyped infectious viruses were used to infect SupT11 (MOI = 0.05)
and THP-1 (MOI = 0.25) cells, thereby creating virus-producing cells (Fig. 1C). The MOI
calculations are based on titers determined by CEM-GXR cells and these MOIs therefore
may not reflect the true MOI on SupT11 or THP-1 cells. The resultant infectious viru­
ses were used to measure viral infectivity in TZM-bl cells, evaluate the packaging of
A3 proteins by western blotting, and analyze the frequency of G-to-A mutations (Fig.
1C). Hereafter, this assay is referred to as pseudo-single cycle infectivity assay (see the“Pseudo-single cycle infectivity assays” section in the Materials and Methods for details).
The susceptibility of hyper-Vif and hypo-Vif HIV-1 to stable A3H hapII protein was
validated in SupT11 cell lines (Fig. S1A). In SupT11-vector cells, hyper-Vif, hypo-Vif, IIIB
Vif, and Vif-deficient HIV-1 had comparable infectivity with TZM-bl cells (Fig. S1A, top
panel). As expected, the infectivity of hypo-Vif and Vif-deficient HIV-1 was restricted in
SupT11-A3H hapII cells because these mutants are unable to degrade A3H hapII protein,leading to packaging of A3H hapII protein into viral particles (Fig. S1A, bottom panel).
The partial degradation of A3H hapII protein by IIIB Vif resulted in moderate inhibitionof IIIB Vif HIV-1 infectivity (Fig. S1A). The infection of hyper-Vif HIV-1 resulted in A3H
hapII degradation, where stable A3H hapII protein is undetectable in the viral particles
and unable to inhibit the Vif mutant (Fig. S1A). Next, to determine whether G-to-A
mutations were introduced into proviral DNA, we recovered proviral DNA from SupT11
cells after infection with each HIV-1 mutant produced from either SupT11-vector or
SupT11-A3H hapII cells and sequenced the pol region. Hypo-Vif and Vif-deficient HIV-1
showed G-to-A mutations preferred by A3H protein (GA-to-AA signature motif; hypo-Vif:
2.8 ± 0.7 mutations/kb and ∆Vif: 3.2 ± 0.8 mutations/kb, respectively) in proviral DNA(Fig. S1B and C). These results are consistent with previous reports demonstrating thesusceptibility of Vif mutants to A3H hapII protein (10).
As shown in Fig. 1D (top panel), hyper-Vif HIV-1, hypo-Vif HIV-1, and IIIB Vif HIV-1(IIIB) produced in THP-1 cells had similar viral infectivity. While Vif did not degrade
A3H protein in THP-1 cells, it was not packaged into viral particles (Fig. 1D, bottom
panel). To examine whether G-to-A mutations were introduced into proviral DNA, wesequenced the pol region of the proviruses from SupT11 cells after infection with eachHIV-1 mutant produced from THP-1 cells. Sequencing data demonstrated that hyper-Vif
HIV-1, hypo-Vif HIV-1, and IIIB Vif HIV-1 had minimal G-to-A mutations preferred by
A3H protein (GA-to-AA signature motif ) in proviral DNA (Fig. 1E and F), indicating that
endogenous A3H protein expressed in THP-1 cells is not involved in HIV-1 restriction. In
contrast, the infectivity of Vif-null HIV-1 was restricted in THP-1 cells and A3G protein
was packaged into viral particles, thereby inducing profound G-to-A mutations (10.3 ±3.5 mutations/kb) (Fig. 1D through F). Most of mutations were in the GG-to-AG signature
motif preferred by A3G protein (80 ± 10%) in proviral DNA (Fig. 1E and F). Taken together,
these results indicate that A3G and perhaps other A3 proteins, but unlikely A3H protein,
contribute to HIV-1 restriction in THP-1 cells.

Development of A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells
A3F protein has a restrictive effect on HIV-1 among A3 family members and is a target
of Vif, in addition to A3G protein, in CD4+ T cell lines and lymphocytes (7, 33–35). To
determine whether A3F protein also reduces HIV-1 infectivity in THP-1 cells, we used
CRISPR to create A3F and A3F/A3G gene knockout cell lines. Two independent subclonesof A3F and A3F/A3G-null THP-1 cells were obtained, as evidenced by the results ofgenomic DNA sequencing and western blotting (Fig. S2).
A3 proteins include single- and double-domain deaminases and are phylogeneticallyclassified into three groups: Z1, Z2, and Z3 domains (3, 4) (Fig. 2A represented in green,orange, and blue, respectively). A3A, A3B carboxy-terminal domain (CTD), and A3G CTD
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proteins are classified as Z1 domains (Fig. 2A, represented in green). Of note, exon 4 of
the A3A gene, exon 7 of the A3B gene, and exon 7 of A3G gene are highly conservedat the nucleotide level (A3A exon 4 and A3B exon 7 have 95% identity; A3A exon 4 andA3G exon 7 have >99% identity; and A3B exon 7 and A3G exon 7 have 95% identity,
respectively). Interestingly, each of these exons has an identical sequence (5´-GAG TGGGAG GCT GCG GGC CA). We therefore designed a guide RNA (gRNA) homologous tothis sequence and used it to delete the entire 125 kbp interval spanning A3A to A3G
genes in THP-1 cells (Fig. 2A, represented in arrows, and Fig. S3). We predicted thatsuccessful Cas9-mediated cleavage would cause one of the following three scenarios:(i) fusion of exon 4 of the A3A gene with exon 7 of the A3B gene (30 kbp deletion);(ii) fusion of exon 7 of the A3B gene with exon 7 of the A3G gene (95 kbp deletion);or (iii) fusion of exon 4 of the A3A gene with exon 7 of the A3G gene (125 kbp
deletion; Fig. 2A). To obtain THP-1 cells lacking expression of A3A to A3G proteins, alentiviral vector expressing gRNA against the target sequence was transduced into THP-1
cells. Finally, two independent subclones (THP-1#11-4 and THP-1#11-7) were obtained,with whole-genome sequencing (WGS) analysis demonstrating an extensive deletion
between A3A exon 4 and A3G exon 7 at the A3 gene locus (Fig. 2B). In THP-1#11-4, six
alleles of the fusion of A3A exon 4 with A3G exon 7 are observed, and each A3A/A3G
hybrid exon had six different insertions or deletions (indels) (Fig. S3). THP-1#11-7 harbors
three alleles of A3A exon 4 and A3G exon 7 fusions (one may be A3A exon 4) with threedifferent deletions (Fig. S3). Although more than 20 potential off-target sites with twoor three nucleotides mismatched with the designed gRNA were predicted, a significant
deletion was only found downstream of the predicted A3G pseudogene harboring 2 bp
mismatched with the target sequence (Fig. S4; potential target sequence in an orange
box and deletions indicated by green dotted lines). In comparison to parental THP-1
cells, these subclones had similar growth capacities under normal cell culture conditions.
Reverse transcription-quantitative PCR (RT-qPCR) analyses demonstrated that A3B to A3G
mRNAs are not detectable in either clone (Fig. 2C). However, A3A mRNA expression
remained detectable in parental THP-1 cells and the two subclones as the A3A promoter
remains intact and potentially functional (Fig. 2A through C). A3A mRNA expression is
known to be upregulated 100- to 1,000-fold in THP-1 cells treated with type I interferon(IFN) (36). To confirm the expression of A3A mRNA and protein in THP-1 cells, parental
THP-1 cell and the respective subclones were cultured in the presence of type I IFN for
6 h, and A3 mRNA and protein expression levels were then analyzed by RT-qPCR andwestern blotting, respectively. In parental THP-1 cells, A3A, A3B, A3F, and A3G mRNAand protein expression levels were increased following IFN treatment (Fig. 2C and D).
In the THP-1#11-4 subclone, A3A mRNA expression is increased following IFN treatment;
however, A3A, A3B, A3C, A3F, and A3G proteins are not detectable, even after IFN
treatment (Fig. 2C and D). Furthermore, A3A to A3G proteins are not detectable in the
THP-1#11-7 subclone under normal cell culture conditions (Fig. 2D). Interestingly, lowlevels of a protein with comparable size to A3A protein are detected in the THP-1#11-7
subclone after IFN treatment (Fig. 2D). Sanger sequence analyses indicated that thisprotein was an A3A and A3G hybrid with a 3 bp deletion (Fig. S3). Collectively, thesedata indicate that the THP-1#11-4 and THP-1#11-7 subclones lack expression of A3A to
A3G proteins under normal cell culture conditions and that clone THP-1#11-4 is a clean
knockout that fails to express functional versions of any of these proteins.

Disruption of A3A to A3G protein expression fully restores the infectivity ofVif-deficient HIV-1 in THP-1 cells
We next determined whether endogenous A3F protein is degraded by Vif in addition toA3G protein. HIV-1 Vif mutants with selective A3 neutralization activities were used forpseudo-single cycle infectivity assays as mentioned above. For example, a Vif4A mutant
harboring 14AKTK17 substitutions (14DRMR17 in IIIB) is susceptible to HIV-1 restriction
activity by A3D and A3F proteins but resistant to the restriction by A3G protein (37–39)(Fig. 3A). We examined the ability of Vif4A to counteract the restriction activity of A3DMonth XXXX Volume 0

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FIG 2 Disruption of the A3A to A3G genes in THP-1 cells. (A) Schematic of the A3 gene at the A3 locus. The A3 family of genes comprises seven members with
one or two Z domains (single- or double-domain deaminases) which belong to three phylogenetically distinct groups shown in green, orange, and blue. Threesites with an identical sequence (5´-GAG TGG GAG GCT GCG GGC CA) in exon 4 of the A3A gene, exon 7 of the A3B gene, and exon 7 of the A3G gene aretargeted by gRNA, as indicated by arrows. The three predicted scenarios are shown. Bar represents 15,000 bp. (B) Mapping of WGS data to the A3 locus. Genomic
DNAs from parental THP-1, THP-1#11-4, and #11-7 cells were subjected to WGS analysis, with an extensive deletion including the A3A–A3G genes observed inof three independent experiments with SD. Statistical significance was determined using the two-sided paired t test. *, P < 0.05 compared to untreated cells.
(D) Representative western blots of three independent experiments. Levels of indicated A3 proteins in whole-cell lysates from cells treated with or without type I
IFN are shown. HSP90 was used as a loading control.

and A3F proteins, although A3D protein could not be detected by western blotting (antiA3D antibodies are unavailable) and it may be inconsequential because its mRNA
expression levels are relatively low in this cell line (26) (Fig. 2C). As our group and others
have shown previously (26, 37, 38, 40), HIV-1 with Vif5A containing five alanine substitu­
tions (40YRHHY44 to 40AAAAA44) is sensitive to the HIV-1 restriction activity of A3G protein
but not A3D and A3F proteins (Fig. 3A). HIV-1 harboring Vif4A5A is susceptible toinhibition by A3D, A3F and A3G proteins (37) (Fig. 3A). VSV-G pseudotyped HIV-1 andthese Vif mutants were used to infect SupT11 derivatives and engineered A3F-null THP-1
cells. First, the susceptibilities of these Vif mutants to A3F and A3G proteins werevalidated in SupT11 cell lines (Fig. S5A). In SupT11-vector cells, Vif-proficient HIV-1 and all
Vif mutants had comparable infectivity in TZM-bl cells (Fig. S5A). As expected, theinfectivity of Vif-deficient HIV-1 and the Vif4A and 4A5A mutants was reduced in SupT11A3F cells as these mutants are unable to degrade A3F protein, thereby leading topackaging of A3F protein in viral particles (Fig. S5A). Further, infection with Vif-deficientHIV-1 or the Vif5A and Vif4A5A mutants resulted in packaging of A3G protein in viralparticles from SupT11-A3G cells in addition to reduced infectivity of these Vif mutants(Fig. S5A). These results are consistent with previous reports demonstrating the suscepti­
bilities of Vif mutants to A3 proteins (26, 37–40).
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THP-1#11-4 and #11-7 clones. (C) RT-qPCR data. Parental THP-1, THP-1#11-4, and #11-7 cells were treated with 500 units/mL type I IFN. Total RNA was isolated
after 6 h. A3 mRNA expression levels were quantified by RT-qPCR and are normalized to TATA-binding protein (TBP) mRNA levels. Each bar represents the averagemBio

FIG 3 Pseudo-single cycle infectivity assays for each HIV-1 mutant in A3-null THP-1 cells. (A) Schematicof the susceptibility of HIV-1 Vif mutants to HIV-1 restriction activity by A3F and A3G proteins. Key aminoacid residues of Vif that determine the susceptibility of HIV-1 IIIB to restriction by A3F and A3G proteins.
−, resistance; +, sensitivity. (B) Representative infectivity of Vif-proficient, Vif-deficient, Vif4A, Vif5A, and
Vif4A5A HIV-1 mutants produced from parental or A3-null THP-1 cells. Top panels show the infectivity ofindicated HIV-1 mutants produced in parental or A3-null THP-1 cells. The amounts of produced viruses
used to infect TZM-bl cells were normalized to p24 levels. Each bar represents the average of four
independent experiments with SD. Data are presented as infectivity relative to Vif-proficient HIV-1 (WT).
Statistical significance was assessed using the two-sided paired t test. *P < 0.05 compared to Vif-proficientHIV-1. Bottom panels are representative western blots of three independent experiments. Levels of
indicated viral and cellular proteins in viral particles and whole-cell lysates are shown. p24 and HSP90
were used as loading controls.
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Pseudo-single cycle infectivity assays were then performed in parental THP-1, A3Gnull, and A3F-null cells using these Vif mutants. Vif-proficient HIV-1 degraded A3F andA3G proteins in THP-1 cells, and lower amounts of these A3 proteins were packaged into
viral particles (Fig. 3B; THP-1 parent). In contrast, Vif-deficient HIV-1 was unable todegrade A3F and A3G proteins, thereby leading to reduced viral infectivity compared to
Vif-proficient HIV-1 (Fig. 3B; THP-1 parent). The infectivity of A3F-susceptible Vif mutants,
Vif4A and Vif4A5A, was lower than that of Vif-proficient HIV-1, indicating that endoge­
nous A3F protein contributes to Vif-deficient HIV-1 restriction in THP-1 cells (Fig. 3B;
THP-1 parent). This finding was supported by results in A3G-null THP-1 cells where Vif4A
mutants are restricted, as observed in parental THP-1 cells (Fig. 3B; THP-1 ∆A3G). The
involvement of endogenous A3G protein in HIV-1 restriction was confirmed in A3G-null
THP-1 cells, as reported (26) (Fig. 3B; THP-1 ∆A3G). To determine whether endogenous
A3F protein contributes to HIV-1 restriction in THP-1 cells, pseudo-single cycle infectivity
assays were performed according to the methods described above in two independent
A3F-null THP-1 clones (Fig. 1C; Fig. S2A and B). Vif-deficient HIV-1 and the Vif5A and
Vif4A5A mutants had reduced infectivity in A3F-null subclones due to the inhibitory
effect of A3G protein (Fig. 3B; THP-1 ∆A3F#1 and #2). However, the infectivity of the Vif4A
mutant was restored to near wild-type levels following disruption of A3F gene expressionin THP-1 cells. These data demonstrate that endogenous A3F and potentially A3D
proteins contribute to Vif-deficient HIV-1 restriction in THP-1 cells, and that Vif degrades
A3F protein and thereby prevents packaging and restriction upon target cell infection.
A3F and A3G proteins are involved in Vif-deficient HIV-1 restriction in THP-1 cells
and are degraded by Vif (26) (Fig. 3B). However, it is unclear whether only these A3
proteins are associated with Vif-deficient HIV-1 restriction in THP-1 cells. To address
this issue, we performed pseudo-single cycle infectivity assays in A3F/A3G-null THP-1
cells using separation-of-function Vif mutants. Although Vif-deficient HIV-1 had greater
infectivity defects in parental, A3G-null, and A3F-null THP-1 cells compared to wild-type
HIV-1 (parent: <10% infectivity, ∆A3G: 30% to 40% infectivity, and ∆A3F: 20% infectivity,
respectively), the infectivity of Vif-deficient HIV-1 was 30% lower in A3F/A3G-null THP-1
cells (Fig. 3B; THP-1 parent, ∆A3G, ∆A3F#1 and #2, and ∆A3F/A3G#1 and #2). On theother hand, the Vif4A, Vif5A, and Vif4A5A mutants had similar infectivity to wild-type
HIV-1 in A3F/A3G-null THP-1 cells (Fig. 3B; THP-1 ∆A3F/A3G#1 and #2). These data indicatethat other A3 proteins, in addition to A3F and A3G proteins, contribute to Vif-deficientHIV-1 restriction in THP-1 cells or that Vif disrupts an additional essential target during
infectious virus production from THP-1 cells.
The universally recognized primary target of Vif is the A3 family of proteins (2, 3,
17, 18). However, Vif-mediated A3 degradation may mask an additional A3-independ­
ent Vif function required for fully infectious virus production. To address this issue,
we constructed two independent A3A-to-A3G-null THP-1 clones (Fig. 2) and character­ized HIV-1 infection using pseudo-single cycle infectivity assays with Vif mutants. As
mentioned above, the disruption of A3F and A3G protein expression results in Vif-deficient HIV-1 having 70% of wild-type HIV-1 infectivity in THP-1 cells (Fig. 3B; THP-1 ∆A3F/
A3G#1 and #2). Remarkably, Vif-deficient HIV-1 and the other Vif mutants have compara­
ble infectivity to Vif-proficient HIV-1 lacking expression of A3A to A3G proteins in THP-1
cells (Fig. 3B; THP-1#11-4 and #11-7). These results indicate that A3 degradation is the
only function of Vif required for fully infectious virus production from THP-1 cells.

A3 proteins restrict HIV-1 infectivity via deaminase-dependent and deami­nase-independent mechanisms in THP-1 cells
Our previous results indicated that A3G protein is the primary source of A3 mutagenesis
in THP-1 cells (26). To further investigate the G-to-A mutation spectra in each A3-null
THP-1 subclone, the pol region was cloned and sequenced from the proviruses used in
the aforementioned infectivity assays. As expected, GG-to-AG mutations are observedin the proviral DNA of Vif mutants lacking A3G neutralization activity (Vif-deficientHIV-1 and Vif5A and Vif4A5A mutants) produced from SupT11-A3G cells (Fig. S5B andMonth XXXX Volume 0

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C; SupT11-A3G). Consistent with a previous report (26), THP-1 expresses A3G protein
capable of mutating A3G-susceptible Vif mutants, including Vif-deficient HIV-1 and Vif5A
and Vif4A5A mutants, as seen in parental THP-1 cells. These GG-to-AG mutations are
not observed in A3G-null THP-1 cells (Fig. 4A and B; THP-1 parent and ∆A3G). Similarly,
GG-to-AG mutations preferred by A3G protein were seen in the proviruses of theA3G-susceptible Vif mutants produced from two independent A3F-null THP-1 cells, with
disruption of A3G protein nearly completely eliminating these mutations in THP-1 cells(Fig. 4A and B; THP-1 ∆A3F#1 and #2, ∆A3F/A3G#1 and #2, #11-4, and #11-7). These
data indicate that A3G protein is the primary source of GG-to-AG mutations in HIV-1
proviruses produced by THP-1 cells.
Although the Vif mutants lacking A3F neutralization activity (Vif-deficient HIV-1 and
Vif4A and Vif4A5A mutants) produced from SupT11-A3F cells have a relatively low
number of G-to-A mutations, the observed G-to-A mutations are predominantly within
the GA-to-AA sequence motif preferred by A3F protein (Fig. S5B and C; SupT11-A3F).
However, A3F-preferred GA-to-AA mutations are not observed in proviruses of A3Fsusceptible Vif mutants produced from parental or A3G-null THP-1 cells, in support ofprior observations (26) (Fig. 4A and B; THP-1 parent and ∆A3G). In addition, fewer GA-toAA mutations are observed in THP-1 cells, even after disruption of A3F protein expression(Fig. 4A and B; THP-1 ∆A3F#1 and #2, ∆A3F/A3G#1 and #2, #11-4, and #11-7). Accordingly,
these results combine to indicate that A3F protein in THP-1 cells is involved in Vifdeficient HIV-1 restriction via a deaminase-independent mechanism.
A3F protein has been shown to inhibit the accumulation of reverse transcription(RT) products (14). To investigate a potential effect on RT, SupT11 cells were infectedwith viruses from the pseudo-single cycle infectivity assays described above, and late
RT (LRT) products were examined by quantitative PCR (qPCR). As expected, all Vif
mutants were decreased in LRT products in comparison to wild-type virus whenthese mutants were produced in parental THP-1 cells and used to infect SupT11 cells(Fig. 4C; THP-1 parent). LRT products of Vif5A and Vif4A mutants were restored tolevels comparable to Vif-proficient HIV-1 following the disruption of A3G or A3F
protein expression in THP-1 cells (Fig. 4C; THP-1 ∆A3G and ∆A3F#1 and #2), indicat­
ing that both A3G and A3F proteins inhibit HIV-1 via a deaminase-independent
mechanism. However, double knockout of A3G and A3F proteins in THP-1 cells did
not increase the LRT products of Vif-deficient HIV-1 compared to those of Vifproficient virus (Fig. 4C; THP-1 ∆A3F/A3G#1 and #2), indicating that other A3proteins, in addition to A3F and A3G proteins, may contribute to the restriction of
HIV-1 in THP-1 cells via a deaminase-independent mechanism or that a separate
protein targeted by Vif blocks the accumulation of RT products. To test thishypothesis, we measured LRT products by infecting SupT11 cells with HIV-1 Vifmutants produced in A3A-to-A3G-null clones. Consistent with the results of thepseudo-single cycle infectivity assays (Fig. 3B), Vif-deficient HIV-1 and other Vif
mutants had comparable levels of LRT products to Vif-proficient HIV-1 lacking
expression of A3A to A3G proteins in THP-1 cells (Fig. 4C; THP-1#11-4 and #11-7).
These data indicate that Vif-mediated A3 degradation is required for fully infectious
virus production from THP-1 to counteract deaminase-dependent and -independent
HIV-1 restriction by A3 proteins.

Transmitted/founder (TF) HIV-1 Vif also only targets A3 family proteins toenable fully infectious virus production from THP-1 cells
As an additional experiment, we examined whether the A3-dependent function of Vif
was present in TF viruses. To address this issue, Vif-proficient and deficient versions of theCH58 TF virus were produced from parental THP-1 and A3A-to-A3G-null cells, with viral
infectivity measured in TZM-bl cells (Fig. 5). Similar to the results observed with IIIB
viruses, Vif-deficient CH58 virus was restricted in parental THP-1 cells; however, thisrestriction is completely abolished by disruption of the A3A to A3G genes (Fig. 5). These
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FIG 4 A3 proteins inhibit Vif-deficient HIV-1 by both deaminase-dependent and -independent mechanisms in THP-1 cells. (A) G-to-A mutations. Average
number of G-to-A mutations in the 564 bp pol gene after infection with hyper-Vif, hypo-Vif, IIIB Vif, or Vif-deficient HIV-1 produced from parental or A3-null THP-1
cells. Each bar depicts the average of three independent experiments with SD. (B) G-to-A mutation profile. Dinucleotide sequence contexts of G-to-A mutations(Continued on next page)
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FIG 4 (Continued)
in the 564 bp pol gene after infection with the indicated viruses produced from indicated cell lines. Each vertical line indicates the location of the dinucleotide
sequence contexts described in the legend within the 564 bp amplicon (horizontal line). (C) Representative LRT quantification data for Vif-proficient, Vif-deficient,
Vif4A, Vif5A, and Vif4A5A HIV-1 mutants produced from each A3-null THP-1 subclone. Data show LRT products of the indicated HIV-1 mutants produced inparental or indicated A3-null THP-1 cells. The amount of produced viruses used to infect SupT11 cells was normalized to p24 levels. LRT products were measured
by qPCR. Each bar represents the average of four independent experiments with SD. LRT products were normalized to the quantity of the CCR5 gene relative to
Vif-proficient HIV-1 (WT). Statistical significance was assessed using the two-sided paired t test. *P < 0.05 compared to Vif-proficient HIV-1 LRT products.

data indicate that TF viruses also utilize a primarily A3-dependent function of Vif during
infectious HIV-1 production from THP-1 cells.

DISCUSSION

FIG 5 Pseudo-single cycle infectivity assays of TF virus molecular clone in A3A-to-A3G-null THP-1 cells.
Infectivity of Vif-proficient and Vif-deficient CH58 viruses. Top panels show the infectivity of Vif-proficientand Vif-deficient HIV-1 produced from parental THP-1, THP-1#11-4, or THP-1#11-7 cells. The amounts
of produced viruses used to infect TZM-bl cells were normalized to p24 levels. Each bar represents theaverage of four independent experiments with SD. Data are represented as relative to Vif-proficientHIV-1 (WT). Statistical significance was assessed using the two-sided paired t test. *P < 0.05 comparedto Vif-proficient HIV-1. The bottom panels are representative western blots of three independent
experiments. The levels of indicated viral and cellular proteins in viral particles and whole-cell lysates
are shown. p24 and HSP90 were used as loading controls.
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Vif-mediated A3 degradation is critical for HIV-1 replication in CD4+ T lymphocytes andmyeloid cells (2, 3, 17, 18). In CD4+ T lymphocytes, at least A3D, A3F, A3G, and A3H(only stable haplotypes) proteins are involved in Vif-deficient HIV-1 restriction, and Vif
is required to degrade A3 enzymes and allow efficient viral replication (2, 3, 17, 18).
However, the degradation of A3 enzymes by Vif during infectious HIV-1 production frommyeloid lineage cells has yet to be fully elucidated. We previously reported that A3G
protein contributes to Vif-deficient HIV-1 restriction in a deaminase-dependent mannerin THP-1 cells (26). Herein, we demonstrate that A3F protein also inhibits Vif-deficientHIV-1 in a largely deaminase-independent manner and that Vif avoids this HIV-1
restriction mechanism by degrading A3F protein (Fig. 3 and 4). Importantly, the resultsof pseudo-single cycle infectivity assays demonstrate that the disruption of A3A to A3G
proteins confers comparable infectivity to wild-type HIV-1 in a Vif-deficient lab-adapted
virus (IIIB) and TF virus (CH58) (Fig. 3 to 5). These results indicate that Vif-mediated A3
degradation is the primary function of Vif during infectious HIV-1 production from THP-1
cells.

Our results demonstrate that A3F and A3G but not A3H proteins restrict Vif-deficientHIV-1 via deaminase-dependent and -independent mechanisms in THP-1 cells (Fig. 1,
3, and 4). In addition to A3F and A3G proteins, our findings indicate that at least one
additional A3 protein is involved in Vif-deficient HIV-1 restriction via a deaminase-inde­
pendent mechanism (Fig. 3 and 4). Accordingly, the remaining four A3 proteins (A3A,
A3B, A3C, and A3D) may contribute to Vif-deficient HIV-1 restriction in a deaminase-inde­
pendent manner in THP-1 cells (Fig. 4). However, A3A and A3B proteins are highly
unlikely to contribute in this manner as A3A mRNA and protein expression levels arevery low or undetectable in THP-1 cells without IFN treatment (Fig. 2C and D). Further,both A3A and A3B proteins are resistant to degradation by HIV-1 Vif (7, 34, 41–43). It istherefore plausible that A3C and A3D proteins contribute to Vif-deficient HIV-1 restrictionin THP-1 cells. An A3C-isoleucine 188 variant is reportedly more active against HIV-1
than a serine 188 variant (44, 45). To ask which A3C variant is expressed by THP-1 cells,
we determined the A3C genotypes of THP-1 cells using cDNA sequencing. These results
demonstrated that the amino acid residue of A3C protein at position 188 is serine.
This result indicates that A3C protein may have a modest effect on Vif-deficient HIV-1
restriction via a deaminase-independent mechanism in THP-1 cells, as shown in priorstudies (44, 45). Similarly, the results of previous studies indicate that A3D protein has aweak effect on Vif-deficient HIV-1 restriction in 293, SupT11, and CEM2n cells (7, 8, 37, 46,
47). Nevertheless, the fact that Vif-deficient HIV-1 has 20% lower infectivity indicates thata synergistic mechanism may enhance the effect of A3 proteins on HIV-1 infectivity (48,49). Further studies are required to fully elucidate the mechanisms underlying the effectof A3 proteins on HIV-1 infectivity.
Similar to CD4+ T lymphocytes, HIV-1 can also target myeloid cells such as mono­cytes and macrophages, and these infections are associated with viral dissemination,
persistence, and latency (50, 51). Accordingly, it is important to understand the role ofrestriction factors, including A3 proteins, in myeloid cells. In monocytes, A3A mRNA levels
are 10 to 1,000 times higher than other A3 mRNA expression levels, and A3A mRNA
expression is reduced by 10- to 100-fold after differentiation into monocyte-derived
macrophages (MDMs) (52–54). In contrast, A3G mRNA expression levels are reduced
approximately 10-fold lower after differentiation of monocytes into MDMs (52, 53). A3FmRNA expression levels are less variable during the differentiation of monocytes into
MDMs (52). Interestingly, suppression of A3A and A3G protein levels by siRNA reportedly
leads to a four- to fivefold increase in p24 production by HIV-1-infected monocytes (53).
As MDMs are generally more sensitive to HIV-1 infection than monocytes, it is highly
likely that A3A and A3G proteins contribute to the lack of susceptibility of monocytes
to HIV-1 infection. However, as previous studies have reported that A3A protein is lessactive against HIV-1 in 293T and SupT11 cell lines (7, 34, 55), further studies are required
to determine the contribution of A3A protein to HIV-1 restriction in monocytes.
In addition to A3A and A3G proteins, A3F and A3H proteins may be involved in
HIV-1 restriction in monocytes. Although A3F mRNA expression levels are essentiallyunchanged during differentiation from monocytes into MDMs (53), A3F mRNA expres­sion levels are comparable to A3G mRNA expression levels (53, 54), indicating thatA3F protein likely contributes to HIV-1 restriction in monocytes. It is possible that only
stable A3H haplotypes and the A3C-I188 variant are associated with HIV-1 restriction inmonocytes. According to previous observations in 293, SupT11, and CEM2n cells (7, 8,
37, 46, 47), A3D protein may modestly contribute to HIV-1 restriction in monocytes. As
A3B mRNA expression levels are relatively low, it is unlikely that this A3B protein inhibits
HIV-1 in monocytes. However, the contribution of A3 proteins other than A3A and A3G
proteins to HIV-1 suppression in monocytes remains unclear, and the antiviral activitiesof these A3 proteins warrant further investigation.
In MDMs, A3A protein appears to be associated with anti-HIV-1 activity as increasing
HIV-1 infectivity has been reported following siRNA knockdown of A3A gene (53, 54).
In addition, HIV-1 replication assays in MDMs using HIV-1 Vif4A and Vif5A mutants
demonstrated that the replication kinetics of both mutants were slower than that
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of the Vif-proficient HIV-1, indicating that A3D, A3F, and A3G proteins contribute toHIV-1 restriction in MDMs (39). However, the effects of A3D and A3F proteins on HIV-1
replication are donor-dependent, likely due to their respective expression levels (39). As
the antiviral activity of A3B, A3C, and A3H proteins has not been reported in MDMs,
further studies are required to address these issues.
Vif is required for HIV-1 replication in CD4+ T lymphocytes and macrophages (2, 3,
17, 18). In the absence of Vif, HIV-1 is attacked by A3 proteins in CD4+ T lymphocytes,
macrophages, monocytes, dendritic cells, and CD4+ T cell lines, and massive G-to-A
mutations accumulate in HIV-1 proviral DNA (7, 8, 10, 15, 23, 26, 39, 56, 57). HIV-1 Vif
recruits A3 proteins into an E3 ubiquitin ligase complex, thereby avoiding the antiviral
activity of these proteins by promoting their degradation through a proteasome-medi­
ated pathway (2, 3, 17, 18). The primary function of Vif has long been posited to be thesuppression of the antiviral activity of A3 proteins. On the other hand, Vif causes G2/M
cell cycle arrest (58–60). As the amino acid residues of Vif responsible for G2/M cell cycle
arrest do not completely match with the amino acid residues required for Vif-mediatedA3 degradation, these functions of Vif may be independent of each other (61–63). In
2016, a functional proteomic analysis identified the PPP2R5 family of proteins, whichfunction as regulators of protein phosphatase 2A, as novel targets of Vif (25). Subse­
quently, studies revealed that Vif induces G2/M arrest by degrading PPP2R5 proteins(60, 64, 65). Vif-induced G2/M arrest has been observed in many cell types, including
293T, SupT11, CEM-SS, and THP-1 cell lines, and primary CD4+ T lymphocytes (25, 61, 63).
However, Vif-mediated G2/M arrest is not required for HIV-1 infection, supporting our
findings that A3 family proteins are the sole essential substrate of Vif during infectious
virus production from THP-1 cells under normal cell culture conditions (Fig. 3 to 5).
It has recently been reported that fragile X mental retardation 1 and diphthamide
biosynthesis 7 are degraded by Vif in CD4+ T lymphocytes (24). Further studies are
required to determine whether a substrate of Vif other than A3 proteins is required forfully infectious HIV-1 production in vivo.
In this study, we revealed an A3-dependent Vif function required for fully infectious
HIV-1 production from THP-1 cells using only one lab-adapted virus (IIIB) and one TF
virus (CH58) (Fig. 3 to 5). However, it is likely that Vif plays additional roles beyond A3
antagonism in some HIV-1 strains. Furthermore, the results of our pseudo-single cycle
infectivity assays do not exclude the possibility that Vif may target non-A3 proteinsrequired for HIV-1 replication. Moreover, we could not exclude the possibility that
immunomodulatory effects may induce additional Vif targets other than A3 proteinsin HIV-1-infected individuals.
In summary, the findings of the studies here demonstrate that the primary target
of Vif is the A3 family of proteins during infectious HIV-1 production from THP-1 cells(i.e., A3G, A3F, and potentially A3C and/or A3D proteins; unlikely A3A, A3B, or A3H hapI
protein). Whether this observation is applicable to primary CD4+ T lymphocytes andmyeloid cells, such as monocytes and macrophages, is important for the development ofantiviral therapies targeting the A3-Vif axis. Such studies may contribute to a functional
cure for HIV-1 by manipulating A3 mutagenesis.

MATERIALS AND METHODS
Cell lines and culture conditions
293T (CRL-3216) was obtained from American Type Culture Collection. TZM-bl (#8129)(66) was obtained from the NIH AIDS Reagent Program (NARP). The creation andcharacterization of the permissive T cell line SupT11 and the SupT11 single clones
stably expressing untagged A3 (SupT11-vector, SupT11-A3F, SupT11-A3G, and SupT11A3H hapII high) have been reported (10, 33). CEM-GXR (CEM-GFP expressing CCR5)
was provided by Dr. Todd Allen (Harvard University, USA) (67). THP-1 was provided
by Dr. Andrea Cimarelli (INSERM, France) (53). The generation and characterization ofMonth XXXX Volume 0

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THP-1 ΔA3G#1 was reported (26). Adherent cells were cultured in DMEM (Wako, Cat#044-29765) supplemented with 10% fetal bovine serum (FBS) (Nichirei, Cat#175012)
and 1% penicillin/streptomycin (P/S) (Wako, Cat# 168-23191). Suspension cells weremaintained in Roswell Park Memorial Institute Medium (RPMI) (Thermo Fisher Scientific,
Cat# C11875500BT) with 10% FBS and 1% P/S.

Genotyping of A3C and A3H genes
Total RNA was isolated from THP-1 by RNA Premium Kit (NIPPON Genetics, Cat#
FG-81250). Then, cDNA was synthesized by Transcriptor Reverse Transcriptase (Roche,
Cat# 03531287001) and used to amplify A3C or A3H gene with the following primers:[A3C outer primers: (5´-GCG CTT CAG AAA AGA GTG GG) and (5´-GGA GAC AGA CCATGA GGC); A3C inner primers: (5´-ACA TGA ATC CAC AGA TCA GAA A) and (5´-CCC CTC
ACT GGA GAC TCT CC); A3H outer primers: (5´-CCA GAA GCA CAG ATC AGA AAC ACG
AT) and (5´-GAC CAG CAG GCT ATG AGG CAA); A3H inner primers: (5´-TGT TAA CAG CCG
AAA CAT TCC) and (5´-TCT TGA GTT GCT TCT TGA TAA T)]. The amplified fragments werecloned into the pJET cloning vector (Thermo Fisher Scientific, Cat# K1231). At least 10
independent clones were subjected to Sanger sequencing (Azenta) and sequence data
were analyzed by Sequencher v5.4.6 (Gene Codes Corporation).

The pLentiCRISPR1000 system was previously described (68). pLentiCRISPR1000-Blast
was generated by restriction digest with BmtI and MluI to excise the P2A-puromycin
cassette. An oligo containing a P2A-blasticidin cassette was purchased from IDT (5´-AGC
GGA GCT ACT AAC TTC AGC CTG CTG AAG CAG GCT GGC GAC GTG GAG GAG AAC CCTGGA CCT ACC GGT ATG GCC AAG CCA CTG TCC CAA GAA GAG TCA ACT CTG ATC GAG
AGG GCC ACT GCA ACC ATT AAT AGC ATT CCC ATC TCT GAA GAC TAT AGC GTA GCT AGT
GCC GCA CTC AGC TCT GAT GGA CGC ATA TTC ACC GGC GTT AAT GTC TAC CAC TTC ACC
GGC GGA CCC TGC GCC GAA CTG GTC GTG CTG GGG ACC GCA GCC GCC GCG GCT GCC
GGG AAT TTG ACG TGC ATT GTT GCA ATA GGC AAC GAG AAT AGG GGC ATC CTG TCA CCTTGC GGC CGG TGT CGG CAA GTG CTG CTG GAC CTG CAC CCC GGC ATC AAG GCC ATA
GTC AAG GAT AGT GAT GGC CAG CCG ACC GCC GTT GGG ATT CGA GAA CTT CTG CCT
TCT GGG TAC GTC TGG GAA GGC TAG) and amplified with the primers (5´-CAA GAC TAGTGG AAG CGG AGC TAC TAA CTT CAG CCT GCT GAA GCA GGC TGG CGA CGT GGA GGA
and 5´-NNN NAC GCG TCT AGC CTT CCC AGA CGT ACC C) using high-fidelity Phusion
polymerase (NEB, Cat# M0530S). The PCR fragment was digested with BmtI and MluI, andligated into the cut pLentiCRISPR1000, producing pLentiCRISPR1000-Blast.

Creation of THP-1 cells disrupting A3 genes
An A3F specific guide for exon 3 was designed (Fig. S2A and C) and evaluated man­ually for specificity to the A3F target sequence via an alignment with the most related
members of the A3 family as described previously (26). Oligos with ends compatible
with the Esp3I sites in pLentiCRISPR1000-Blast were purchased from IDT [ΔA3F gRNA:(5´-CAC CGG TAG TAG TAG AGG CGG GCG G) and (5´-CCA TCA TCA TCT CCG CCC GCCCAA G)]. The targeting construct was generated by annealing oligos and cloned byGolden Gate ligation into pLentiCRISPR1000-Blast. A guide with a common sequenceamong A3A exon 4, A3B exon 7, and A3G exon 7 was designed (Fig. 2A) and oligos
with ends compatible with the Esp3I sites in pLentiCRISPR1000 (68) were purchasedfrom IDT [PanZ1 gRNA: (5´-CAC CGT GGC CCG CAG CCT CCC ACT C) and (5´-GAA CGAGTG GGA GGC TGC GGG CCA C)]. The targeting construct was generated by annealingoligos and cloned by Golden Gate ligation into pLentiCRISPR1000 (68). All constructs
were confirmed by Sanger sequencing (Azenta) and sequence data were analyzed by
Sequencher v5.4.6 (Gene Codes Corporation).
For transduction, VSV-G pseudotyped virus was generated by transfecting 2.5 µgof the pLentiCRISPR1000 or pLentiCRISPR1000-Blast targeting construct along with
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Construction of pLentiCRISPR-Blast

1.67 µg of pΔ-NRF (HIV-1 gag, pol, rev, tat genes) (69) and 0.83 µg of pMD.G (VSV-G)
expression vectors using TransIT-LT1 (Takara, Cat# MIR2306) into 293T cells. At 48 h
post-transfection, viral supernatants were harvested, filtered with 0.45 µm filters (Merck,Cat# SLHVR33RB), and concentrated by centrifugation (26,200 × g, 4°C, 2 h). Then, viralpellets were resuspended in 10% FBS/RPMI and incubated with cells for 48 h. Fortyeight hours later, cells were placed under drug selection in 10% FBS/RPMI containing
1 µg/mL puromycin (InvivoGen, Cat# ant-pr) or 6 ng/mL blasticidin (InvivoGen, Cat#
ant-bl). Single-cell clones were isolated by the limiting dilution of the drug-resistantcell pool and expanded. The expression levels of A3F protein in THP-1 ΔA3F#1 and #2
and THP-1ΔA3F/A3G#1 and #2 cells were confirmed by western blotting (see “Western
blot” section). To confirm indels in the A3F target sequence of the selected clones,
genomic DNA was isolated by DNeasy Blood & Tissue Kits (Qiagen, Cat# 69504) andamplified with Choice-Taq DNA polymerase (Denville Scientific, Cat# CB4050-2) using
primers (5´-GCT GAA GTC GCC CTT GAA TAA ACA CGC and 5´-TGT CAG TGC TGG CCC CG).
The amplified PCR products were cloned into the pJET cloning vector (Thermo Fisher
Scientific, Cat# K1231) and subjected to Sanger sequencing (Azenta). To confirm indels in
the A3A, A3B, and A3G target sequences of the selected clones (THP-1#11-4 and #11-7),
genomic DNA was isolated by DNeasy Blood & Tissue Kits (Qiagen, Cat# 69504) andsubjected to whole-genome sequencing (Macrogen). The WGS data were enrolled in the
NCBI BioSample database and the respective accession numbers are SAMN35719796 forparental THP-1, SAMN35719797 for THP-1#11-4, and SAMN35719798 for THP-1#11-7. The
sequencing data were aligned by Isaac aligner (iSAAC-04.18.11.09). Off-target sites wereanalyzed by Cas-OFFinder (http://www.rgenome.net/cas-offinder/). For further analysis
of indels between A3A and A3G genes, genomic DNAs from THP-1#11-4 and #11-7
were amplified using primers (5´-GGG GCT TTC TGA AAG AAT GAG AAC TGG GC and5´-CAG CTG GAG ATG GTG GTG AAC AGC C). The amplified PCR products were cloned
into the pJET cloning vector (Thermo Fisher Scientific, Cat# K1231) and subjected toSanger sequencing (Azenta). All sequence data were analyzed by Sequencher v5.4.6(Gene Codes Corporation). To assess the expression levels of A3 mRNAs and proteins,
THP-1 parent, #11-4, and #11-7 were incubated in 10% FBS/RPMI including 500 units/mL
IFN (R&D Systems, Cat# 11200-2) for 6 h. Then, cells were harvested and subjected
to RT-qPCR (see “RT-qPCR” section) (Fig. 2C) and western blotting (see “Western blot”
section) (Fig. 2D).

Pseudo-single cycle infectivity assays
Vif-proficient and Vif-deficient (X26 and X27) HIV-1 IIIB C200 proviral expression constructs
have been reported (70). HIV-1 IIIB C200 mutants with hyper- (H48 and 60EKGE63) andhypo-functional (V39) Vifs have been reported (10). An HIV-1 IIIB C200 Vif 5A mutant(40AAAAA44) has been described (26). HIV-1 IIIB C200 Vif 4A (14AKTK18) and 4A5A
(14AKTK18 and 40AAAAA44) mutants were created by digesting pNLCSFV3-4A, and −4A5A
proviral DNA construct [(37); kindly provided by Dr. Kei Sato, University of Tokyo,
Japan] at SwaI and SalI sites and cloned into pIIIB C200 proviral construct. The proviral
expression vector encoding full-length TF virus, CH58 (NARP, #11856) was obtained fromthe NARP. The creation of Vif-deficient CH58 mutant has been described previously (71).
HIV-1 single-cycle assays using VSV-G pseudotyped viruses were performed as
described previously (23, 26) (Fig. 1C). 293T cells were cotransfected with 2.4 µg of
proviral DNA construct and 0.6 µg of VSV-G expression vector using TransIT-LT1 reagent(Takara, Cat# MIR2306) into 293T cells (3 × 106). Forty-eight hours later, supernatants
were harvested, filtered (0.45 µm filters, Merck, Cat# SLHVR33RB), and used to titrate
on 2.5 × 104 CEM-GXR reporter cells for MOI determinations. GFP+ cells were measured
using a FACS Canto II (BD Biosciences) and the data were analyzed using FlowJo softwarev10.7.1 (BD Biosciences). One or 5 × 106 target cells were infected with an MOI of 0.05 (for
SupT11 derivatives) or 0.25 (for THP-1 derivatives) and washed with phosphate-bufferedsaline (PBS) twice at 24 h postinfection and then incubated for an additional 24 h.
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quantified by p24 ELISA (ZeptoMetrix, Cat# 0801008) and used to infect 1 × 104 TZM-blcells (1 or 2 ng of p24). At 48 h postinfection, the infected cells were lysed with aBright-Glo luciferase assay system (Promega, Cat# E2650) and the intracellular luciferase
activity was measured by a Synergy H1 microplate reader (BioTek) or Centro XS3 LB960
microplate luminometer (Berthold Technologies).

Quantification of LRT products
Viruses were produced by infecting VSV-G pseudotyped virus into THP-1 cells as
described above (see “Pseudo-single cycle infectivity assays” section) and the resulting
viral particles were quantified by p24 ELISA (ZeptoMetrix, Cat# 0801008). The viralsupernatants including 20 ng of p24 antigen were used for infection into SupT11 cells.
At 12 h postinfection, cells were harvested and washed with PBS twice. Then, total
DNA was isolated by DNeasy Blood & Tissue Kits (Qiagen, Cat# 69504) and treated with
RNase A (Qiagen, Cat# 19101) according to the manufacturer’s instruction. Following
DpnI digestion, 50 ng of DNA was used to amplify LRT products and CCR5 gene withthe following primers: LRT forward: (5´-CGT CTG TTG TGT GAC TCT GG) and LRT reverse:(5´-TTT TGG CGT ACT CAC CAG TCG); CCR5 forward: (5´-CCA GAA GAG CTG AGA CAT
CCG) and CCR5 reverse (5´-GCC AAG CAG CTG AGA GGT TAC T). qPCR was performed
using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Cat# 4367659) andfluorescent signals from resulting PCR products were acquired using a Thermal Cycler
Dice Real Time System III (Takara). Finally, each LRT product was represented as values
normalized by the quantity of the CCR5 gene (Fig. 4C).

Cells were harvested and washed with PBS twice. Then, total RNA was isolated by RNA
Premium Kit (NIPPON Genetics, Cat# FG-81250) and cDNA was synthesized by Transcrip­
tor Reverse Transcriptase (Roche, Cat# 03531287001) with random hexamer. RT-qPCR
was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific,
Cat# 4367659). Primers for each A3 mRNA have been reported previously (72, 73): A3A
forward: (5´-GAG AAG GGA CAA GCA CAT GG) and A3A reverse: (5´-TGG ATC CAT CAA GTG
TCT GG); A3B forward: (5´-GAC CCT TTG GTC CTT CGA C) and A3B reverse: (5´-GCA CAG
CCC CAG GAG AAG); A3C forward: (5´-AGC GCT TCA GAA AAG AGT GG) and A3C reverse:(5´-AAG TTT CGT TCC GAT CGT TG); A3D forward: (5´-ACC CAA ACG TCA GTC GAA TC)
and A3D reverse: (5´-CAC ATT TCT GCG TGG TTC TC); A3F forward: (5´-CCG TTT GGA CGC
AAA GAT) and A3F reverse: (5´-CCA GGT GAT CTG GAA ACA CTT); A3G forward: (5´-CCG
AGG ACC CGA AGG TTA C) and A3G reverse: (5´-TCC AAC AGT GCT GAA ATT CG); A3H
forward: (5´-AGC TGT GGC CAG AAG CAC) and A3H reverse: (5´-CGG AAT GTT TCG GCTGTT); TATA-binding protein (TBP) forward: (5´-CCC ATG ACT CCC ATG ACC) and TBP reverse:(5´-TTT ACA ACC AAG ATT CAC TGT GG). Fluorescent signals from resulting PCR products
were acquired using a Thermal Cycler Dice Real Time System III (Takara). Finally, each A3mRNA expression level was represented as values normalized by TBP mRNA expression
levels (Fig. 2C).

Hypermutation analyses
Hypermutation analyses were performed as previously described (23, 26, 45). Genomic
DNAs containing HIV-1 proviruses were recovered by infecting viruses produced inderivatives of THP-1 or SupT11 cells into SupT11 using DNeasy Blood & Tissue Kits
(Qiagen, Cat# 69504). Following DpnI digestion, the viral pol region was amplified bynested PCR with outer primers (876 bp) [(5´-TCC ART ATT TRC CAT AAA RAA AAA)and (5´-TTY AGA TTT TTA AAT GGY TYT TGA)] and inner primers (564 bp) [(5´-AAT ATTCCA RTR TAR CAT RAC AAA AAT) and (5´-AAT GGY TYT TGA TAA ATT TGA TAT GT)].
The resulting 564 bp amplicon was subjected to pJET cloning. At least 10 independ­
ent clones were Sanger sequenced (Azenta) for each condition and analyzed by theMonth XXXX Volume 0

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HIV sequence database (https://www.hiv.lanl.gov/content/sequence/HYPERMUT/hyper­
mut.html). Clones with identical mutations were eliminated.

Western blot
Western blotting for cell and viral lysates was performed as described previously (23, 26,74). Cells were harvested, washed with PBS twice, and lysed in lysis buffer [25 mM HEPES(pH7.2), 20% glycerol, 125 mM NaCl, 1% Nonidet P40 (NP40) substitute (Nacalai Tesque,
Cat# 18558-54)]. After quantification of total protein by protein assay dye (Bio-Rad, Cat#
5000006), lysates were diluted with 2× SDS sample buffer [100 mM Tris-HCl (pH 6.8), 4%
SDS, 12% β-mercaptoethanol, 20% glycerol, 0.05% bromophenol blue] and boiled for 10
min. Virions were dissolved in 2× SDS sample buffer and boiled for 10 min after pelleting
down using 20% sucrose (26,200 × g, 4°C, 2 h). Then, the quantity of p24 antigen wasmeasured by p24 ELISA (ZeptoMetrix, Cat# 0801008).
Proteins in the cell and viral lysates (5 µg of total protein and 10 ng of p24 anti­gen) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF)
membranes (Millipore, Cat# IPVH00010). Membranes were blocked with 5% milk in PBS
containing 0.1% Tween 20 (0.1% PBST) and incubated in 4% milk/0.1% PBST contain­
ing primary antibodies: mouse anti-HSP90 (BD Transduction Laboratories, Cat# 610418,
1:5,000); rabbit anti-A3B (5210-87-13, 1:1,000) (75); rabbit anti-A3C (Proteintech, Cat#
105911-1-AP, 1:1,000); rabbit anti-A3F (675, 1:1,000) (76); rabbit anti-A3G (NARP, #10201,
1:2,500); rabbit anti-A3H (Novus Biologicals, NBP1-91682, 1:5,000): mouse anti-Vif (NARP,#6459, 1:2,000); mouse anti-p24 (NARP, #1513, 1:2,000). Subsequently, the membranes
were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies:
donkey anti-rabbit IgG-HRP (Jackson ImmunoResearch, 711-035-152, 1:5,000); donkey
anti-mouse IgG-HRP (Jackson ImmunoResearch, 715-035-150, 1:5,000). SuperSignal West
Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat# 34095) or Super
signal atto (Thermo Fisher Scientific, Cat# A38555) was used for HRP detection. Bands
were visualized by the Amersham Imager 600 (Amersham).

Statistical significance was performed using a two-sided paired t test (Fig. 1D, 2C, 3B, 4C,
5, S1A, and S5A). GraphPad Prism software v8.4.3 was used for these statistical tests.
ACKNOWLEDGMENTS
We would like to thank Haruyo Hasebe, Kimiko Ichihara, Kazuko Kitazato, Otowa
Takahashi, and all Ikeda lab members for technical assistance. We also would like tothank Drs. Todd Allen, Andrea Cimarelli, and Kei Sato for sharing reagents.
This study was supported in part by AMED Research Program on Emerging andRe-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108494 to
Terumasa Ikeda); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa
Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa
Ikeda); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham
Nasser); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa
Ikeda); Takeda Science Foundation (to Terumasa Ikeda); Mochida Memorial Founda­tion for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Founda­tion (to Terumasa Ikeda); Shin-Nihon Foundation of Advanced Medical Research (toTerumasa Ikeda); Waksman Foundation of Japan (to Terumasa Ikeda); an intramural grantfrom Kumamoto University COVID-19 Research Projects (AMABIE) (to Terumasa Ikeda);
Intercontinental Research and Educational Platform Aiming for Eradication of HIV/AIDS(to Terumasa Ikeda); International Joint Research Project of the Institute of Medical
Science, the University of Tokyo (to Terumasa Ikeda); SPP1923 and the Heisenberg
Program of the German Research Foundation (SA 2676/3-1; SA 2676/1-2) (to Daniel
Sauter); as well as the Canon Foundation Europe (to Daniel Sauter).
Month XXXX Volume 0

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Statistical analyses

Research Article

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Contributions from the Harris lab were supported by NIAID R37-AI064046 anda Recruitment of Established Investigators Award from the Cancer Prevention andResearch Institute of Texas (CPRIT RR220053). R.S.H. is an Investigator of the Howard
Hughes Medical Institute and the Ewing Halsell President’s Council Distinguished Chair.
The authors declare that they have no competing interests.
AUTHOR AFFILIATIONS
1Division of Molecular Virology and Genetics, Joint Research Center for Human Retrovirus
Infection, Kumamoto University, Kumamoto, Japan
2
Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan3
Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Ismailia,
Egypt
4
Department of Biochemistry and Structural Biology, University of Texas Health San
Antonio, San Antonio, Texas, USA
5
Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio,
Texas, USA
6
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota,
Minneapolis, Minnesota, USA
7
Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA
8
Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital
Tübingen, Tübingen, Germany
AUTHOR ORCIDs
Terumasa Ikeda http://orcid.org/0000-0003-2869-9450
Adam Z. Cheng http://orcid.org/0000-0001-6277-9433
Daniel Sauter http://orcid.org/0000-0001-7665-0040
Reuben S. Harris http://orcid.org/0000-0002-9034-9112

FunderGrant(s)Author(s)Japan Agency for Medical Research and Develop­
ment (AMED)

JP21fk0108574

Hesham Nasser

Japan Agency for Medical Research and Develop­
ment (AMED)

JP21fk0108494,
JP22fk0410055

Terumasa Ikeda

MEXT | Japan Society for the Promotion of Science 22K07103(JSPS)

Terumasa Ikeda

MEXT | Japan Society for the Promotion of Science 22K16375(JSPS)Hesham Nasser

Takeda Science Foundation (TSF)

Terumasa Ikeda

MEXT | Japan Society for the Promotion of Science(JSPS)

Terumasa Ikeda

Mochida Memorial Foundation for Medical andPharmaceutical Research (公益財団法人 持田記
念医学薬学振興財団)

Terumasa Ikeda

Naito Foundation (内藤記念科学振興財団)

Terumasa Ikeda

Shinnihon Foundation of Advanced Medical
Treatment Research (公益財団法人 新日本先進医療研究財団)

Terumasa Ikeda

Waksman Foundation of Japan

Terumasa Ikeda

Canon Foundation in Europe (CFE)

Daniel Sauter

HHS | NIH | National Institute of Allergy and
Infectious Diseases (NIAID)
Month XXXX Volume 0

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R37-AI064046

Reuben S. Harris

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FUNDINGmBio

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AUTHOR CONTRIBUTIONSTerumasa Ikeda, Conceptualization, Investigation, Supervision, Validation, Writing –original draft, Writing – review and editing | Ryo Shimizu, Conceptualization, Investiga­
tion, Validation, Writing – review and editing | Hesham Nasser, Investigation, Validation,
Writing – review and editing | Michael A. Carpenter, Investigation, Validation, Writing– review and editing | Adam Z. Cheng, Investigation, Validation, Writing – review andediting | William L. Brown, Investigation, Validation, Writing – review and editing |Daniel Sauter, Investigation, Validation, Writing – review and editing | Reuben S. Harris,
Investigation, Validation, Writing – review and editing
ADDITIONAL FILES
The following material is available online.
Supplemental Material
Figure S1 (mBio00782-23-s0001.tif). Pseudo-single cycle infectivity assays for eachHIV-1 mutant in SupT11 cells stably expressing stable A3H haplotype.
Supplemental figure legends (mBio00782-23-s0002.docx). Legends to Fig. S1 to S5.
Figure S2 (mBio00782-23-s0003.tif). Development of A3F and A3F/A3G-null THP-1 cells.
Figure S3 (mBio00782-23-s0004.tif). Sequence analysis of flanking region targeted bygRNA in THP-1#11-4 and #11-7.
Figure S4 (mBio00782-23-s0005.tif). Deletions around predicted A3G pseudogene.
Figure S5 (mBio00782-23-s0006.tif). Pseudo-single cycle infectivity assays for eachHIV-1 mutant in SupT11 cells stably expressing A3.

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