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1
POLYMORPHISMS AND SPLICE VARIANTS INFLUENCE THE ANTIRETROVIRAL ACTIVITY OF
HUMAN APOBEC3H
Ariana Harari*, Marcel Ooms*, Lubbertus C. F. Mulder, Viviana Simon
Departments of Medicine and Microbiology,
Emerging Pathogens Institute
Mount Sinai School of Medicine, New York, NY 10029, USA
* These authors contributed equally
Running head: Mutations and Alternative Splicing in human APOBEC3H
Abstract: 264 words
Text including legends and references: 7370 words (including references and legends)
Figures: 7
Address correspondence to:
Dr. V. Simon, Mount Sinai School of Medicine,
One Gustave L. Levy Place, Box 1090,
New York City, NY 10029, USA
Tel: (212) 241 8388
Fax: (212) 849 2643
Email: [email protected]
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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.01665-08 JVI Accepts, published online ahead of print on 22 October 2008
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ABSTRACT
Human APOBEC3H belongs to the APOBEC3 family of cytidine deaminases that
potently inhibit exogenous and endogenous retroviruses. The impact of single nucleotide
polymorphisms (SNP) and alternative splicing on the antiretroviral activity of human
APOBEC3H is currently unknown. In this study, we show that APOBEC3H transcripts
derived from human peripheral blood mononuclear cells are polymorphic in sequence and
subject to alternative splicing. We found that APOBEC3H variants encoding a SNP cluster
(G105R, K121D and E178D, hapII-RDD) restricted HIV-1 more efficiently than wild-type
APOBEC3H (hapI-GKE). All APOBEC3H variants tested were resistant to HIV-1 Vif, the
viral protein that efficiently counteracts APOBEC3G/3F activity. Alternative splicing of
APOBEC3H was common and resulted in variants with distinct C-terminal regions and
variable antiretroviral activity. Splice variants of hapI-GKE displayed a wide range of
antiviral activities, whereas similar splicing events in hapII-RDD resulted in proteins that
uniformly and efficiently restricted viral infectivity (>20-fold). Site-directed mutagenesis
identified G105R in hapI-GKE and D121K in hapII-RDD as critical substitutions leading
to an average additional 10-fold increase in antiviral activity. APOBEC3H variants were
catalytically active and, similarly to APOBEC3F, favored a GA dinucleotide context. HIV-1
mutagenesis as mode of action for APOBEC3H is suggested by the decrease of restriction
observed with a cytidine deaminase domain mutant and the inverse correlation between G-
to-A mutations and infectivity.
Thus, the anti-HIV activity of APOBEC3H seems to be regulated by combination of
genomic variation and alternative splicing. Since prevalence of hapII-RDD is high in
populations of African descent, these findings raise the possibility that some individuals
may harbor effective as well as HIV-1 Vif resistant intracellular antiviral defense
mechanisms.
ABBREVIATIONS
APOBEC: Apolipoprotein B mRNA editing enzyme, catalytic polypeptide
A3: APOBEC3
HIV-1: Human immunodeficiency virus type 1
PBMC: peripheral blood mononuclear cells
SNP: single nucleotide polymorphism
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INTRODUCTION
APOBEC3H is a member of the APOBEC3 family of cytidine deaminases, some of
which possess strong anti-HIV-1 activity (e.g., APOBEC3G/3F) (3, 9, 13, 16, 21, 34). HIV-1’s
ability to replicate in human cells depends on the expression of the viral protein Vif, which
efficiently mediates the degradation of several APOBEC3 members in the producer cell (9, 13,
21).
APOBEC3H messenger RNA has been detected in several human tissues (e.g., peripheral
blood mononuclear cells, [PBMC], liver, skin, ovary and testis (19, Ohainle, 2006 #1628)).
APOBEC3H lacks the cytidine deaminase domain (CDA1) that mediates RNA binding,
homodimerization and virion encapsidation of APOBEC3G (14, 26). In contrast to the strong
Vif-independent HIV-1 restriction exerted by the rhesus macaque APOBEC3H, the human
protein seems to be limited in its antiretroviral activity (10, 28). Protein expression levels of
human APOBEC3H and that of the rhesus homologue differ greatly upon transfection into
mammalian cells (10, 28), suggesting that the lack of potency of human APOBEC3H is a
reflection of insufficient expression and/or protein stability in the producer cell, rather than a
lack of enzymatic activity. Indeed, human APOBEC3H displayed cytidine deaminase activity
comparable to its rhesus homologue in a bacterial mutator assay (28). Moreover, APOBEC3H
has been reported to cause hypermutation in both Hepatitis B virus (19) and in Human
Papillomavirus genomes (33) suggesting the presence of enzymatic activity in mammalian
systems.
Comparison between human and rhesus sequences revealed that rhesus APOBEC3H
protein is 210 amino acid long whereas the human homologue is shorter due to a premature
translation termination codon. Repairing this stop codon resulted in a human APOBEC3H
protein variant which was well expressed in mammalian cells and displayed HIV-1 Vif
independent antiretroviral activity (11). A similar activity profile was observed when expression
of the short human APOBEC3H variant was optimized using a CMV intron A containing
expression vector (11).
In this study, we report that multiple, distinct APOBEC3H variants with antiviral activity
are present in PBMC from healthy donors. Specifically, we identified a cluster of three non-
synonymous single nucleotide polymorphisms (SNP) which in conjunction with a specific splice
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variant confer strong, HIV-1 Vif resistant antiretroviral activity. This restriction correlated with
the introduction of G-to-A mutations in HIV-1 proviruses in a GA dinucleotide context.
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MATERIAL AND METHODS
Amplification of APOBEC3H transcripts: Human PBMC were obtained by Ficoll (GE
Healthcare) density centrifugation from 12 HIV-1 negative anonymous blood donors (Mount
Sinai School of Medicine Blood Bank). Cells were cryopreserved in liquid nitrogen until total
cellular RNA was extracted using Qiagen RNA extraction kit. RNA was reverse transcribed with
Superscript II (Invitrogen) and random hexamers. APOBEC3H variants were amplified with
PicoMax DNA polymerase (Stratagene) using primers 5' - AAC GCT CGG TTG CCG CCG
GGC GTT TTT TAT TAT GGC TCT GTT AAC AG and 5' - TCT TGA GTT GCT TCT TGA
TAA T. PCR products were cloned using StrataClone kit (Stratagene) as specified by the
manufacturers’ instructions. Six to fourteen clones per donor were sequenced using BigDye
Terminator v3.1 reagents and analyzed on an ABI PRISM 3730xl (Agencourt Bioscience Corp.).
Sequences were manually edited and aligned using DNASTAR and Bioedit software packages.
Plasmids used for HIV-1 production: Replication competent full-length molecular clone NL4-3
Vif mutant SLQ144AAA (NL4-3 FSLQ) has previously been described (24). It lacks the
required motif to bind to Elongin C, which is part of the E3 ligase complex Cullin5/ Elongin B/C
needed for APOBEC3 degradation (13).
Plasmid HIV-1 gag-pol (pCRV1/gag-pol) (17), the packagable HIV-1 RNA genome encoding
Tat, Rev, Vpu and GFP (pV1/hrGFP), the G protein from vesicular stomatitis virus (pHCMV
VSV-G) and the Vif expression plasmid pCRV1-Vif have been described previously (30).
APOBEC3 expression plasmids: Six of the most common APOBEC3H variants (hapI-GKE and
hapII-RDD; SV-182, SV-183, SV-200) were subcloned into the mammalian expression vector
pTR600 (15). We chose pTR600 because its CMV intron A improves expression of the inserted
transgene (15). APOBEC3H variants were amplified from StrataClone plasmids (see
amplification of APOBEC3H transcripts) using Pfu polymerase (Stratagene) and the following
primers: 5’- GAT CAA GCT TCG ATG GAT TAC AAG GAT GAC GAC GAT AAG ATG GCT
CTG TTA ACA GCC GAA AC (FLAG-tag sequence in italic) and 5'- TAA TAC GAC TCA
CTA TAG GG. Upon restriction enzyme digestion and ligation into pTR600, the cloned inserts
were verified by sequencing.
Site-directed mutagenesis of APOBEC3H: Plasmid pTR600-hapI-GKE and pTR600-hapII-RDD
(both SV-183) were used as template for site-directed APOBEC3H mutagenesis. We used
standard overlap PCR techniques to introduce mutations at positions 56, 105, 121 and 178.
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Mutation E56A is located in the deaminase active site (CDA) and has been shown to abolish
catalytic activity in other APOBEC3 enzymes. Introduction of the correct mutation into the
cloned fragments was confirmed by sequencing.
Culture of cell lines: HEK 293T and TZM-bl reporter cells were maintained in Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100U/ml
penicillin/streptomycin. TZM-bl cells were provided by J.C. Kappes and X. Wu through the
AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes
of Health, NIH Reagent program.
Transfection of HEK 293T: Viral stocks were obtained by transfection of HEK 293T cells using
4ug/ml polyethylenimine (PEI; Polysciences, Inc.). pTR600-FLAG-APOBEC3G, pTR600-
FLAG-hapI-GKE, FLAG-hapII-RDD expression vectors (range 50 to 1000ng) were co-
transfected with NL4-3 WT, NL4-3 Vif mutant SLQ144AAA molecular clones (500ng) or
irrelevant plasmids (500ng) in 24-well tissue culture plates.
HIV-1 vector particles were generated by transfecting HEK 293T cells with plasmids
pCRV1/gag-pol, the packagable HIV-1 RNA genome pV1/hrGFP and pHCMV VSV-G in a
5:5:1 ratio (30). To measure APOBEC3H and Vif functions, cells were co-transfected with this
plasmid mixture and additional plasmids expressing the amino-terminally FLAG-tagged
APOBEC3H variants with pCRV1empty or pCRV1/Vif wild-type.
In all transfections, the culture media was replenished after 12 hours. Supernatants were
harvested two days after transfection, clarified by centrifugation and used to infect TZM-bl
reporter cells.
Assessment of viral infectivity. TZM-bl reporter cells, which carry an HIV-1 Tat responsive
beta-galactosidase indicator gene under the transcriptional control of the HIV-1 LTR, were used
to assess the infectivity of viral stocks produced by transfection in the presence and absence of
the different FLAG-APOBEC3H variants or FLAG-APOBEC3G. TZM-bl cells were infected in
triplicate with 20ol cell-free viral supernatants in 96-well plates. Beta-galactosidase activity was
quantified 48 hours after infection using chemiluminescent substrate (Tropix, Perkin-Elmer), as
previously described (30).
Western blotting of cell lysates. Cells were lysed in 1% SDS, 50 mM Tris HCL pH 8.0, 150 mM
NaCl, 5 mM EDTA, supplemented with EDTA-free protease inhibitor cocktail (Roche) 48 hours
post-transfection. Proteins were separated on 10% or 4-12% gradient polyacrylamide SDS gels
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(Invitrogen), transferred to PVDF membranes (Pierce) and probed with c-FLAG M2 monoclonal
antibody (Sigma) for FLAG-APOBEC3G and FLAG-APOBEC3H variants. Membranes were
subsequently incubated with horseradish peroxidase conjugated secondary antibodies and
developed with SuperSignal West Pico (Pierce). After stripping with 0.2 M NaOH for 10
minutes, membranes were probed with c-GAPDH (Sigma) to ensure equal protein loading.
For quantification of protein expression, western blots were developed as described
above and analyzed using the Fujifilm intelligent lightbox LAS-3000 and Image Reader LAS-
3000 software. Signals were detected at super sensitive settings with 10 sec. increments. Only
the non-saturated signals were quantified using ImageGauge 4.0 software and used to calculate
protein expression levels.
APOBEC3H-driven HIV-1 mutagenesis: Viral stocks were generated by transfecting NL4-3 WT
(500ng) and pTR600-FLAG-APOBEC3H variants (50ng), pTR600-FLAG-APOBEC3G (50ng)
or pTR600 (50ng) in HEK 293T cells. Culture media was replaced the next day and supernatants
were harvested 36 hours later. TZM-bl cells were infected in 24-well tissue culture plates with
DNase I (Invitrogen) treated viral stocks. 12 hours post infection, the cells were extensively
washed with PBS and genomic DNA was extracted using DNeasy DNA isolation kit (Qiagen).
To assess the frequency of mutations in the proviral genome a 1905 nucleotide long region of pol
(HXB2: nucleotides 2928-4833) was amplified by PCR and cloned using StrataClone kit as
previously described (24). DNA sequencing was performed by Agencourt Biosciences using
BigDye Terminator v3.1 reagents. RT sequences (600bp) were manually edited and aligned
using DNASTAR and Bioedit software packages. The frequency of G-to-A mutations and the
dinucleotide context of the mutations were analyzed with the Hypermut program (29).
Statistical Analysis: Prism software (version 4.0 GraphPad Software) was used to perform all
statistical tests. P values are two-sided and values < 0.05 were considered to be significant.
Accession numbers for APOBEC3H: The reference accession ID for the APOBEC3H HAPII-
RDD (rs numbers) are rs139292 (F15N), rs139293 (R18L), rs139294 (synonymous G-to-C
nucleotide substitution at position 129), rs139297 (R105G), rs139298 (K121E), rs139299
(K121N), rs139302 (E178D). Representative APOBEC3H cDNA sequences were submitted to
Genbank (accession numbers pending).
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RESULTS
The current human APOBEC3H mRNA sequence information is based on some 30
different cDNA clones submitted to GenBank/dbEST (accessed May 2008). The APOBEC3H
gene comprises seven gt-ag introns and four alternatively spliced mRNAs are predicted to
encode functional proteins (63, 182, 183 and 200 amino acids, see AceView;
http://www.ncbi.nlm.nih.gov /IEB/ Research/Acembly/index.html?human and (32)).
APOBEC3H variant NM_181773 (Ensembl ID ENS00000100298) has served as wild-
type reference (11) and will be referred to as hapI-GKE (in agreement with the nomenclature
used by OhAinele et al, (27)). For clarity purposes, we named the different APOBEC3H splice
variants based on the length of the predicted protein (e.g., SV-182, SV-183, SV-200).
APOBEC3H is polymorphic in sequence.
Nine APOBEC3H SNPs (one synonymous [+129C/G, T43T], two single codon deletions
[F14N, F15N] and six non-synonymous [R18L, G37H, G105R, K121E/N, S140G, E178D]) are
listed in the Single Nucleotide Polymorphism database at NCBI (www.ncbi.nlm.nih.gov/
projects/SNP).
We amplified, cloned and sequenced APOBEC3H transcripts derived from PBMC cDNA
of 12 anonymous blood donors. Six to 14 APOBEC3H clones were analyzed for each donor
(total: 106 clones).
We detected 6 SNPs at previously published polymorphic positions in our dataset (Fig
1A displays the exon location of the mutations; the rs numbers are listed in the Methods section).
APOBEC3H alleles encoding GKE at position 105, 121 and 178 respectively were amplified
from 10 of 12 donors indicating that Haplotype 1 is common (Fig. 1B). APOBEC3H transcripts
encoding a cluster of three substitutions G105R, K121D and E178D were found in 6 of the 12
donors (Figure 1B). Haplotype II (HapII-RDD with “RDD” standing for the substitutions at
position 105, 121 and 178, respectively) was seen in 3 of the 12 donors (Fig. 1B). Deletion of
asparagine at position 15 (F15N) with or without substitution R18L (haplotype III and IV,
respectively, Fig. 1B) was observed in combination with cluster RDD in four independent
clones, derived from three different donors (donors D1, D4, D8). The synonymous mutation
+129C (residue T43) was detected in 6 donors, always in conjunction with hapII-RDD, hapIII or
hapIV.
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Two donors harbored exclusively G105R/K121D/E178D APOBEC3H transcripts
(donors P6, P7) and 6 donors only wild-type APOBEC3H suggesting that these individuals are
homozygous for hapII-RDD or hapI-GKE. Mixtures of wild-type and mutant transcripts (hapII-
RDD: D11; hapIII: D1, D8; haplotype IV: D4) were recovered from the PBMC of the remaining
4 donors (Fig. 1B). Our data indicate that APOBEC3H is polymorphic in sequence with hapI-
GKE and hapII-RDD being commonly represented in the 12 donors studied.
The International HapMap Consortium project provides information on the frequency of
SNPs in four populations of diverse ethnicities (7, 12) and lists distribution for some of the
APOBEC3H SNPs analyzed in this study (www.hapmap.org). Of note, the aspartic acid (D) at
position 121 in hapII-RDD was encoded by mutations at the first and third position of the triplet
(lysine K: AAG to D: GAC), whereas the SNP database lists the polymorphisms separately (N:
AAC or E: GAG) resulting in distinct residues. Fig. 1C lists the allele’s frequencies for SNPs
105R, 121E and 178D in four different populations. For example, SNP G105R is highly
prevalent in Sub-Saharan Africans (e.g., 93% of Yoruba [HapMap-YRI] encode G105R) but less
often observed in others groups (e.g., only 39% of European [HapMap-CEU] and 31% of Asians
[HapMAp-HCB and JPT] encode G105R). Data for K121E and E178D reveal a similar ethnic
bias supporting our observation that G105R/K121D/E178D occur as cluster rather than as
isolated substitutions (Fig.1C).
APOBEC3H transcripts are subject to alternative splicing.
Estimates suggest that half of all human genes are subjected to alternative splicing,
thereby generating transcriptome diversity in a cell-type or tissue-specific manner (23).
Figure 2A depicts the four alternatively spliced APOBEC3H forms (SV-183, SV-182,
SV-200 and SV-154) that were found in at least two different blood donors. All transcripts with
the exception of SV-154 share exons 2, 3 and 4 with SV-182 lacking only a glutamine in the
second from last position in exon 5. In contrast, skipped and/or cryptic exons in SV-200 (cryptic
exon 4b) and SV-154 (skipped exon 4 and cryptic exon 4b) result in 19 and 15 distinct amino
acids, at the C-terminus, respectively (see Fig. 2B for the predicted protein sequences of each of
these transcripts). A limited number of matches are present in databases for SV-182 (9 cDNA
clones) and SV-200 (2 cDNA clones), while SV-154 has not been described (see AceView
http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index. html?human).
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An assortment of two or three APOBEC3H transcripts was present in the PBMC of all
donors. SV-183 and SV-182 were detected in all donors in contrast to some of the alternative
splice forms (e.g., SV-200: 6/12 donors; SV-154: 3/12 donors; Fig 2C). In summary, alternative
splicing of APOBEC3H transcripts occurred frequently in vivo and resulted in proteins with
variable C-terminal regions.
The antiviral activity of hapII-RDD APOBEC3H variants is superior to hapI-GKE.
We next investigated the impact of genomic sequence variation on APOBEC3H function.
We used two experimental approaches which differ in the manner by which HIV-1 Vif is
delivered. In Approach 1 HIV-1 WT or Vif mutant (SLQ144AAA) were provided in cis by full-
length molecular clone NL4-3. In Approach 2, HIV-1 Vif was supplied in trans allowing for Vif
complementation independently of the HIV-1 genome. In both systems, the infectivity of viral
particles generated in the presence of human APOBEC3H variants was assessed on the TZM-bl
reporter cell-line.
1) APOBEC3H activity using full-length HIV-1: The infectivity of HIV-1 WT and mutant
(SLQ144AAA) Vif viruses produced in the presence of the APOBEC3H variants was compared
to the infectivity of viruses made in the presence of APOBEC3G or in the absence of any
APOBEC3 (Fig. 2A). The hapI-GKE SV-183 inhibited HIV-1 (WT Vif) infectivity by more than
20-fold (Fig. 3A), which is comparable to the decrease obtained with APOBEC3G (hapI-GKE
[NL4-3]: 3.8% +/- 2.9 versus APOBEC3G [NL4-3]: 9.3% +/- 0.4; P = ns, paired T-test). HapII-
RDD SV-183 showed a significantly higher antiretroviral activity compared to APOBEC3G
(SV-183 of hapII-RDD [NL4-3]: 1.5% +/- 0.5 versus APOBEC3G [NL4-3]: 9.3% +/- 0.4; P =
0.006, paired T-test).
The restriction exerted by hapI-GKE and hapII-RDD APOBEC3H variants was
comparable for HIV with and without functional Vif which stands in contrast to the Vif-mediated
rescue of viral infectivity observed for viruses produced in the presence of APOBEC3G (e.g.
compare NL4-3 WT with NL4-3 Vif mutant SLQ144AAA in Fig 3A).
A 100-fold reduction of NL4-3 Vif mutant SLQ144AAA infectivity was observed for
hapII-RDD; a level of restriction that was comparable to the one induced by APOBEC3G (SV-
183 of hapII-RDD [NL4-3 SLQ144AAA]: 1.07% +/- 0.1 versus APOBEC3G [NL4-3
SLQ144AAA]: 0.36% +/- 0.6; P = 0.06, paired T-test). In this system hapI-GKE and hapII-RDD
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APOBEC3H both function as HIV-1 Vif resistant antiviral restriction factors with the hapII-
RDD being more active.
Western blots of cell lysates revealed that hapI-GKE and hapII-RDD variants differed in
the level of protein expression. Only hapII-RDD proteins were well expressed upon transfection
(Fig. 3B, right part). However, when co-transfected with HIV-1 NL4-3, the accumulation of all
APOBEC3H proteins, including hapI-GKE variants, was greatly enhanced (Fig. 2B, right part).
These findings indicate that mutations within APOBEC3H (e.g., G105R/K121D/E178D cluster)
as well as HIV-1 itself can, independently, increase and/or stabilize human APOBEC3H
expression.
2) APOBEC3H activity using HIV vector system: To confirm the HIV-1 Vif independent
nature of the APOBEC3H restriction, we generated HIV-1 vector-derived VSV-G pseudotyped
viral particles with and without Vif proteins in the presence of APOBEC3H variants and
measured their infectivity (Fig. 4). Under these experimental conditions, viral infectivity was
reduced by two-fold in the presence of hapI-GKE and 5- to 20-fold by hapII-RDD (SV-183 and
SV-200). This restriction was completely independent of the presence of HIV-1 Vif, which
stands in good agreement with the findings observed with the full-length Vif SLQ144AAA
mutant virus. The activity of APOBEC3G was comparable between the two systems:
APOBEC3G action was HIV-1 Vif sensitive as shown by the >100-fold reduction of infectivity,
which is rescued by addition of HIV-1 Vif (e.g., 10% of the level observed in the absence of
APOBEC3, also compare controls in Fig 2A and Fig. 3A).
Western blotting of the cell lysates revealed that the expression levels of hapII-RDD
variants were comparable in the presence or absence of HIV-1 Vif in contrast to APOBEC3G
which is readily degraded by HIV-1 Vif (Figure 4B). HapI-GKE was, however, poorly expressed
in the presence of viral vectors complemented with HIV-1 Vif or empty control plasmid (e.g.,
compare expression in Fig. 3B and 4B). These data suggest that the antiretroviral activity of
APOBEC3H is completely independent of the presence of HIV-1 Vif and not only in its ability
to bind to the Elongin C component of the Cullin5 E3 ligase. Furthermore, it seems likely that
either the absence of some of the non-structural genes in the vector-derived viruses or a different
ratio between genomic RNA and GagPol in the producer cell can account for the different degree
of APOBEC3H accumulation and antiviral activity seen with this approach.
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The influence of alternative splicing on the antiviral activity depends on the genetic background
of APOBEC3H
Since alternative splice forms were frequently detected in PBMC, we tested next whether
splice variants of hapI-GKE and hapII-RDD differed in their activity against HIV-1. We tested
three isoforms (SV-182, SV-183, SV-200) for hapI-GKE and hapII-RDD.
SV-182 and SV-200 in hapI-GKE were less active than SV-183 (SV-182: 14.02% +/-
6.9, P= 0.018; SV-200: 19.46% +/- 3.1, P= 0.001; paired T-test, Fig 3A). In contrast, SV-182 and
SV-200 in the hapII-RDD background displayed increased activity compared to SV-183 (e.g.,
50- to 100-fold reductions of infectivity, Fig. 3A, 5A). Of note, hapII-RDD/SV-200 reduces viral
infectivity to the levels observed for APOBEC3G with the notable difference that infectivity was
not recued by a functional Vif (e.g., HIV-1 SLQ144AAA in Fig. 3A, and viral vectors +/- Vif in
Fig 4A).
Serial dilutions of the different APOBEC3H splice variants confirmed the activity
differences between wild-type and hapII-RDD splice variants (Fig. 5A). Protein expression of
serially diluted APOBEC3H variants showed that hapII-RDD variants were expressed to higher
levels than the hapI-GKE ones (Fig. 4B). Interestingly, the level of expression of hapI-GKE
variants was comparable for all three splice forms despite clear differences in antiviral activity
(compare Fig. 4A with Fig. 4B). Thus, although APOBEC3H protein expression levels might be
relevant for anti-HIV-1 activity, other features (e.g., cellular localization) are likely to have an
equally important role.
Figure 5C illustrates in more details the impact of alternative splicing on antiviral activity
relative to the allelic context. Extended serial dilutions of APOBEC3H expression plasmids
(50ng-1000ng) reveal dramatically different activity profiles for hapI-GKE and hapII-RDD SV-
200 (far right panels in Fig 5C) with SV-200 of hapII-RDD achieving suppression levels
comparable to those of APOBEC3G (far left panel and light grey lines in Fig 4). Taken together,
these findings suggest that alternative splicing regulates the antiviral function of both
APOBEC3H haplotypes with opposite effects.
Requirements for activity in wild-type and mutant genomic APOBEC3H context
To determine the amino acid substitutions within the SNP cluster (G105R/K121D/
E178D) that are responsible for differences between hapI-GKE and hap-II-RDD, we constructed
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a panel of site-directed mutants containing the naturally occurring amino acid substitutions at
position 105, 121 and 178 in different combinations (all SV-183, Fig. 6A). As in previous
experiments, the infectivity of WT NL4-3 viruses produced in the presence of 100ng
APOBEC3H variants was assessed by infection of TZM-bl. We choose this intermediate
concentration of APOBEC3H expression plasmid in order to have optimal discrimination in the
lower range of the assay (see also serial dilutions in Fig. 5C).
Residues in two distinct positions (105R, 121K) proved to be relevant for the
antiretroviral activity of hapI and hapII APOBEC3H (Fig. 6A). Introduction of 105R into hapI-
GKE resulted in a protein that was 10-fold more active than the original protein (NL4-3
infectivity in the presence of hapI-RKE: 1.2% +/-1.3 versus hapI-GKE: 22.0% +/-7.2).
Conversely, replacement of arginine at position 105 by glycine in hapII-RDD increased
infectivity approximately 3-fold (NL4-3 in the presence of hapII-RDD: 7.5%+/-4.1 versus hapII-
GDD: 27.1%+/- 9.1) yielding levels of infectious particle release comparable to hapI-GKE (Fig.
6A).
In hapII, the reversion from aspartic acid to lysine at position 121 (hapII-RKD) resulted
in a 4-fold increase of antiretroviral activity compared to the naturally occurring hapII-RDD
(NL4-3 infectivity in the presence of hapII-RKD: 1.9%+/-1.3 versus hapII-RDD: 7.5%+/-4.1).
Substitutions at position 178 in either haplotype did not improve the potency (compare hapI-
GKD and hapII-RDE to their corresponding parental proteins, Fig. 5A).
Since expression of hapI-GKE was far inferior to hapII-RDD, we speculated that
mutations 105R may result in protein stabilization thereby leading to enhanced activity. In
agreement with our previous findings (e.g. Fig 3B, 5B), the expression of hapII-RDD was 5-fold
higher than hapI-GKE (Fig 6B). However, expression of hapI-RKE, carrying the 105R
substitution from hapII, was 10 times higher than that of the natural variant hapI-GKE. In
contrast, hapII protein expression was destabilized by introduction of hapI residues at position
105 and 178 (hapII-GDD and RDE).
Taken together these findings suggest that the activity of both haplotype I and II is
suboptimal and can be improved by specific substitutions from the other haplotype. The
combination of 105R and 121K (hapI-RKE and hapII-RKD, Fig 6A/B) resulted in stably
expressed proteins with, in average, 10-fold higher antiviral activity.
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APOBEC3H variants deaminate HIV-1
Since the ability to act as mutator of retroviral genomes is a hallmark of APOBEC3
proteins, we next investigated whether APOBEC3H variants introduce G-to-A changes into
HIV-1 upon infection of target cells. TZM-bl reporter cells infected with NL4-3 viral stocks
produced in the presence of APOBEC3H variants (50ng, hapI-GKE and hapII-RDD, SV183 and
SV-200), an active site mutant (100ng, E56A, hapII-RDD) or APOBEC3G (50ng). Genomic
DNA of these infected cells was used to amplify, clone and sequence the HIV-1 RT region.
In parallel measurement of the infectivity of each viral stock revealed that catalytic site
mutant HapII-E56A failed to restrict HIV-1 (e.g, NL4-3 infectivity 94.2% +/-1.8, Fig 7A). Under
these experimental conditions, APOBEC3G reduced infectious NL4-3 virus production by 2-fold
(52%+/- 5.01), while infectivity rates in the presence of APOBEC3H ranged between 21.3%
(SV-200 of hapII-RDD) and 90.4% (SV-200 of hapI-GKE, Fig 7A).
We analyzed 6 to 11 individual RT clones for each infection (total: 59 clones, 35,400
nucleotides, Fig 7B/D) and calculated the overall frequency of mutations, the percent of G-to-A
mutations as well as the favored dinucleotide context in which they occurred (e.g., GG versus
GA). Overall, the APOBEC3H variants introduced mostly G-to-A mutations (Fig. 7B).
Infectivity correlated with the degree of mutagenesis detected in the proviral sequences. For
example, SV-200 of hapII-GKE induced the most mutations (frequency of any mutation 0.79%,
only G-to-A mutations 0.68%, Fig. 7B) and restricted HIV-1 best (Fig 7A). Similarly, SV-200 of
hapI-GKE is the least active of the APOBEC3H variants and the frequency of mutations
associated with this viral stock was very low. Indeed, with an overall mutation frequency of
0.09%, SV-200 of hapI-GKE was comparable to the virus alone (e.g., “no APOBEC3 control”
0.09%, Fig. 7B) and to the active site mutant E56A (e.g., 0.1%, Fig 7B). Lastly, the frequency of
G-to-A mutations correlated inversely with the infectivity (Fig. 7C).
The majority of G-to-A mutations introduced by APOBEC3H occurred in a GA
dinucleotide context which contrasted with APOBEC3G which clearly favored a GG
dinucleotide context (compare Fig. 7C and 7D). A similar preference has been reported for
human APOBEC3F (20, 35) and rhesus macaque APOBEC3H (28).
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DISCUSSION
Human APOBEC3H is evolutionarely distinct from the other six human APOBEC3
family members: it resembles the 3’ region of mouse APOBEC3 more closely than any
APOBEC3 domain of human origin (8, 28). Interestingly, the mouse APOBEC3 is as active
against HIV-1 as is the human APOBEC3G but, unlike APOBEC3G, it is fully resistant to HIV-
1 Vif (22).
Human APOBEC3H is the least studied of the single domain cytidine deaminases, which
generally exert only modest anti-HIV activity (9, 13). We thought, therefore, to investigate
whether sequence variation and/or splicing events may increase its antiviral activity. We started
by analyzing the frequency of APOBEC3H SNP and splice variants in PBMC, a cell population
known to express APOBEC3H (11, 19, 28). Here we describe APOBEC3H to be polymorphic in
sequence and subject to alternative splicing (Fig. 1 and 2).
We generated a panel of the most commonly detected haplotypes (hapI-GKE and hapII-
RDD) and splice variants (SV-183, SV-182, SV-200) and tested them for antiretroviral activity
and expression (Fig. 3A, 3B). Four of the six APOBEC3H variants inhibited the infectivity of
HIV-1 20- to 100-fold in a Vif-independent manner. Our findings indicate that all splice variants
of hapII-RDD were well expressed and active against HIV-1 (Fig. 2A). Thus, hapII-GKE
APOBEC3H variants are highly active HIV-1 Vif resistant antiviral proteins, which mimics
mouse APOBEC3 anti-HIV-1 properties (4, 6).
Frequency, pattern, function and relevance of alternative splicing of most human
APOBEC3 enzyme remains unknown but it is tempting to speculate that alternative splicing of
cryptic exons could provide functional diversity and/or control. Human APOBEC3B has been
reported to have two splice variants, both of them expressed in human liver but the shorter form
lacks activity against Hepatitis B (5). In mice, two APOBEC3 splice variants display similar
activity against HIV-1 (6) and in cats alternative read-through splicing generates APOBEC3CH
(A3C-H fusion protein, (25)).
In this study, we find that splice variants modulate the antiviral activity of APOBEC3H.
Splice events that lead to the replacement of the carboxy terminal region of the protein were
frequent (Fig. 2B) and the majority of donors harbored combinations of two or three different
variants. By using PBMC to amplify APOBEC3H transcripts, our current data do not
discriminate which cell populations express the different splice variants. It is conceivable that
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cell-type specific alternative splicing events lead to the accumulation of certain variants which,
depending on the genotype (hapI-GKE versus hapII-RDD), could be highly active or largely
defective (compare wild-type SV-200 with SNP SV-200 in Fig. 5C) with respect to their ability
to inhibit HIV-1 infectivity.
APOBEC3 enzymes restrict HIV-1 through editing and non-editing mechanisms
(reviewed by (9, 13, 18). The degree of mutagenesis observed was in excellent agreement with
the reduction of viral infectivity observed for the specific APOBEC3H variants. Although this is
only a correlation, the catalytic mutant provides compelling evidence of the causal relationship
between deamination and viral restriction (Fig 7B). These findings for human APOBEC3H
resemble those relative to rhesus APOBEC3H, which are catalytically active and display a strong
preference for a GA dinuclotide context (28).
Studies of natural history cohorts have reported associations between non-synonymous
SNPs in APOBEC3G as well as in Cullin-5 genes (1, 2). Individuals differ in their susceptibility
to infection and time to AIDS disease progression and future studies will establish whether
individuals with these SNPs in the APOBEC3H gene are more resistant to HIV-1/AIDS disease.
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NOTE ADDED DURING REVISION
Reports by Ohainle et. al. (27) and Tan et al. (31) were published as this manuscript was
under revision. We attempted to integrate the different nomenclatures to facilitate understanding.
Haplotype I represents the wild-type reference sequence and is referred to as hapI-GKE.
Haplotype 2 represents APOBEC3H alleles containing a cluster of three SNP
(G105R/K121D/E178D) and is named hapI-RDD. “GKE” or “RDD” stand for the amino acids
found at positions 105, 121 and 178 of APOBEC3H.
ACKNOWLEDGMENTS
We thank C. Linscheid and C. Seibert for technical assistance. P. Bieniasz, L.
Chakrabarti, C. Cheng-Mayer for helpful discussions and T. Ross for kindly providing pTR600
plasmid.
This work was supported by NIH grants R01 AI064001 (V.S.) and R21 AI073213
(L.C.F.M.). V.S. is a Sinsheimer Scholar (Alexandrine and Alexander L. Sinsheimer Fund).
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FIGURE LEGENDS
Figure 1: Sequence diversity of APOBEC3H transcripts derived from PBMC of healthy
blood donors.
A: Summary of single nucleotide polymorphisms (SNP) detected in APOBEC3H transcripts
derived from human PBMC. Location of the five non-synonymous mutations (black square) and
the one synonymous mutation (square with black and white stripes) are indicated. CDA denotes
the cytidine deamination site of the enzyme.
B: The APOBEC3H alleles with the different mutations at position 15, 18, 105, 121 and 178 are
listed together with the frequency of detection in 12 PBMC donors analyzed in this study.
C: Summary of the genetic diversity of APOBEC3H SNP detected in human populations for
SNP 105R, 121E and 178D (based on HapMap database). The details of each genotype reveal
differences in the mutation frequency for Utah residents with ancestry from Europe (CEU), Han
Chinese (HCB), Japanese in Tokyo, Japan (JPT) and Yoruba from Ibadan, Nigeria (YRI).
Figure 2: Alternative splicing of APOBEC3H transcripts derived from PBMC of healthy
blood donors.
A: The four most frequently detected APOBEC3H splice variants are shown each with its
corresponding splice pattern. SV denotes splice variant with the number reflecting the length of
the protein (e.g., SV-182 encodes 182 residues). SV-182 and SV-183 have previously served as
reference. The most pronounced changes were noted for SV-200 and SV-154 due to the skipping
of exon 4 and/or the usage of the cryptic exon 4b. The schematic representation has been adapted
from AceView (www.ncbi.nlm.nih.gov)
B: Alternative splicing introduced variation in the 3’ end of APOBEC3H. The four alternative
proteins (SV-182, SV-183, SV-200, SV-154) are depicted with their distinct carboxyl terminal
regions being underlined.
C: The assortment of APOBEC3H splice variants within each donor is shown. Between 6 and 14
independent clones were analyzed for each donor (total: 106 clones). The majority of donors
harbored at least three different APOBEC3H splice variants with SV-182 and SV-183 being
detected in every donor.
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Figure 3: Antiretroviral activity profiles of naturally occurring APOBEC3H variants using
full length HIV-1
A: HIV-1 wild-type (NL4-3, 500ng, closed box) and Vif mutant SLQ144AAA (NL4-3〉SLQ,
500ng, open box) viral stocks were produced by transfection in the presence of six different
APOBEC3H variants (250ng), APOBEC3G (250ng) or empty pTR600 plasmid (250ng). Viral
infectivity was assessed by infection of TZM-bl reporter cells. Results were normalized using the
no-APOBEC3 controls as reference and plotted as percent relative infectivity. HapI-GKE and
HapII-RDD refer to the APOBEC3H haplotype. Results represent the mean +/- standard
deviation (SD) of TZM-bl infections performed in triplicates from at least two independent
transfection experiments. A3H stands for APOBEC3H.
B: APOBEC3H expression levels in the absence and presence of NL4-3 HIV-1 were assessed by
western blotting of transfected HEK 293T cell lysates. APOBEC3H variants are FLAG-tagged at
the amino terminus. The predicted molecular weight for the normal splice form of APOBEC3H
is 23.5 kDa. Detection of GAPDH served as protein loading control.
Figure 4: Antiretroviral activity profiles of naturally occurring APOBEC3H variants using
HIV-1 vectors
A: VSV-G pseudotyped, HIV-1 viral vectors were produced in the presence of APOBEC3H
variants and APOBEC3G with and without HIV-1 Vif expression plasmids. Viral infectivity was
quantified by TZM-bl reporter cell infection. Results were normalized using the no-APOBEC3
controls as reference and plotted as percent relative infectivity. Results represent the mean +/-
SD of TZM-bl infections performed in triplicates from two independent transfection
experiments.
B: The expression levels of APOBEC3H variants and APOBEC3G in the presence of viral
vectors complemented HIV-1 Vif or empty plasmid were assessed by western blotting of
transfected HEK 293T cell lysates.
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Figure 5: Alternative splicing impacts the antiviral activity of APOBEC3H
A: Co-transfection of NL4-3 (500ng) and serial dilutions (50, 100, 250ng) of the six
APOBEC3H plasmids demonstrate the different impact of alternative splicing on hapI-GKE (left
panel) and hapII-RDD (right panel). Results represent the mean +/- SD of TZM-bl infections
performed in triplicates from a representative transfection experiment.
B: Western blot analysis of the lysates of cells used for the production of viruses in the presence
of increasing APOBEC3H concentrations (50, 100, 250ng) as depicted in Fig. 3C. g-FLAG
monoclonal antibody was used to probe for FLAG-APOBEC3H expression. Detection of
GAPDH served as protein loading control.
C: Transfection of serial dilutions (50-1000ng) of APOBEC3H plasmids demonstrate higher
antiviral potency of SV-200 hapII-RDD compared to SV-183 and SV-200 of hapI-GKE (black
open and closed squares). As a reference the relative activity of APOBEC3G against NL4-3 and
NL4-3 Vif mutant SLQ144AAA is shown in grey symbols in each of the plots. Error bars
represent SD of TZM-bl infections performed in triplicates form a representative transfection
experiment.
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Figure 6: Identification of mutations essential for antiviral activity of APOBEC3H
A: The impact of single mutations on antiretroviral activity was tested by co-transfecting the
different APOBEC3H site-directed mutants (100ng) with NL4-3 (500ng). Viral infectivity was
quantified by TZM-bl reporter cell infection. Results were normalized using the no-APOBEC3
controls as reference and plotted as percent relative infectivity. Results represent the mean +/-
SD of TZM-bl infections performed in triplicates from four independent transfection
experiments. The cartoons on the right illustrate the panel of mutants with the letters in the boxes
symbolizing the amino acids at position 105, 121 and 178 in hapI-GKE (white box) and hapII-
RDD (dark box) backgrounds.
G: glycine; K: lysine, E: glutamic acid, R: arginine, D: aspartic acid
B: The expression levels of APOBEC3H site-directed mutants were assessed by western blotting
of transfected HEK 293T cell lysates. The FUJI intelligent light box LAS-300 system was used
to quantify protein expression in a dynamic manner. The bar graph shows protein expression
normalized to the signal captured for hapI-GKE (first lane). g-FLAG monoclonal antibody was
used to probe for FLAG-APOBEC3H expression. Detection of GAPDH served as protein
loading control.
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Figure 7: APOBEC3H variants introduce G-to-A mutations in HIV-1
A: Infectivity of NL4-3 viral stocks produced in the presence of hapI-GKE and hapII-RDD
variants (50ng) was assessed on TZM-bl reporter cells. hapII-RDD E56A is a catalytic site
mutant (SV-183). Results represent the mean +/- SD of TZM-bl infections performed in
triplicates from one representative experiment.
B: TZM-bl cells were infected with viral stocks produced in the presence of APOBEC3H
variants, the catalytic site mutant E56A and APOBEC3G. Genomic DNA was extracted and a
portion of RT was amplified, cloned and sequenced (6 to 14 clones for each infection). The
number of mutations was calculated relative to the total number of sequenced nucleotides for
each infection.
C: Infectivity and the relative frequency of G-to-A mutations correlate inversely. HapI-GKE/SV-
200, hapII-RDD-E56A (deaminase site mutant) and pTR600/no A3 control cluster closely in the
upper left corner.
D: APOBEC3 mutagenesis depends on the dinucleotide context (e.g. GG versus GA). The
number of G-to-A mutations per clone are plotted for SV-183 and SV-200 of hapI-GKE and
hapII-RDD. APOBEC3G favors a GG dinucleotide context whereas APOBEC3H prefers a GA
dinucleotide context.
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5
E178DG105R K121D
4
F15N R18L
3 2
Human APOBEC3H
X
CDA1
nt129 g/cNon-syn:
Syn:
Exon #
Harari et al.,
Figure 1
A
B15 18 105 121 178
Abbr. usedMutation in A3H
hapI-GKE
hapII-RDD
hapIV
83% (10/12)
25% (3/12)
8% (1/12)
17% (2/12)R
L
FF
R D D
R D
DR
D
D
L
LN
N G K E
hapIII
Frequency in
PBMC donorsC
CEU HCB YRI
105
121
178
R
E
D
JPT
39 31 31 93
39
39
31
36
31
32
93
88
A3H
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Harari et al.,
Figure 2
A
C
B
SV-154
SV-183
Exon 2 Exon 3 Exon 4 Exon 5Exon 1
Exon 4bSV-182
SV-200
SV-183 MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENK
KKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHD
HLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVD
HEKPLSFNPYKMLEELDKNSRAIKRRLERIKQS*
SV-182MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENK
KKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHD
HLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVD
HEKPLSFNPYKMLEELDKNSRAIKRRLERIKS*
SV-200MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENK
KKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHD
HLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVD
HEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV*
SV-154MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENK
KKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHD
HLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPDSRGTCAGSLHGYIV*
D1
D2
D3
D4
D5
D6
D7
D8
D9
D1
0
D1
1
D1
2
0
20
40
60
80
100 SV-182
SV-183
SV-200
SV-154
other
Donors
Sp
lice
Va
ria
nts
(%
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Harari et al.,
Figure 3
hapI-GKE hapII-RDD Controls
SV-1
83
SV-1
82
SV- 2
00
SV-1
83
SV-1
82
SV- 2
00
APOBEC
3G
pTR60
00.1
1
10
100
NL4-3 NL4-3 SLQ144AAA
Rela
tive I
nfe
cti
vit
y(p
erc
en
t o
f n
o A
3)
A
hapIIhapI hapIIhapI
SV
-182
SV
-183
SV
-200
SV
-183
SV
-182
SV
-200
SV
-182
SV
-183
SV
-200
SV
-183
SV
-182
SV
-200
NL4-3Control plasmid
A3H
c-FLAG
c-GAPDH
B
ACCEPTED
on February 19, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Harari et al.,
Figure 4
A Controls
SV-1
83
SV-2
00
SV-1
83
SV-2
00
APOBEC
3G
pTR60
0
HIV-1 Vif No HIV-1 Vif
Rela
tive I
nfe
ctv
ity
(perc
en
t o
f n
o A
3)
hapI-GKE hapII-RDD GKE
SV
-183
SV
-200
SV
-183
SV
-200
A3G
pT
R600
RDD GKE
SV
-183
SV
-200
SV
-183
SV
-200
A3G
pT
R600
RDD
+ HIV-1 Vif No HIV-1 Vif
c-FLAG
c-HIV-1 Vif
c-GAPDH
A3H
Vif
B
0.1
1
10
100
A3G
hapI hapII hapI hapII
ACCEPTED
on February 19, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
A3H
A3G
BA
Harari et al.,
Figure 5
C
c-GAPDH
c-FLAG
SV-183 SV-182 SV-200
hapI hapII hapI hapII hapI hapII
APOBEC3H splice variants
hapI-GKE
Rela
tive I
nfe
cti
vit
y(p
erc
en
t o
f n
o A
3)
SV-183
Plasmid concentration (ng)
hapI-GKE
Rela
tive I
nfe
cti
vit
y(p
erc
en
t o
f n
o A
3)
010
020
030
00.1
1
10
100
SV-183
SV-182
SV-200
A3H plasmid concentration
(ng)
NL4-3
hapII-RDD
010
020
030
00.1
1
10
100
SV-183
SV-182
SV-200
SV-200 APOBEC3G
hapI-GKE hapII-RDD NL4-3
NL4-3 SLQ144AAA
0250
500750
10000.1
1
10
100
0250
500750
10000.1
1
10
100
0250
500750
10000.1
1
10
100
0250
500750
10000.1
1
10
100
ACCEPTED
on February 19, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Relative Infectivity (percent of no A3)
Residue position
1 1 0 1 0 0
105 121 178
G K
R K E
G D E
G K
E
D
R D
G D D
R K D
R D E
D
105 121 178
hapI-GKE
hapI-RKE
hapII-RDD
hapI-GKD
hapI-GDE
hapII-GDD
hapII-RKD
hapII-RDE
pTR600
hap I-GK
E
hap I-RK
E
hap I-GD
E
hap I-GK
D
pTR600
hap II-R
DD
hap II-G
DD
hap II-R
KD
hap II-R
DE
pTR600
0
5
10
15
Fo
ld d
iffe
ren
ce
in e
xpre
ssio
n
17.6 %
27.1 %
7.5 %
100 %
29.5 %
20.6 %
22.0 %
1.9 %
1.2 %
Harari et al.,
Figure 6
A
B
c-FLAG
(A3H)
c-GAPDH
ACCEPTED
on February 19, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Harari et al.,
Figure 7
A CB
Infe
ctiv
ity
(% o
f n
o A
3)
0.0
0.5
1.0
1.5
G-to-A
any other substitution
Fre
qu
en
cy o
f m
uta
tio
n (
%)
0
20
40
60
80
100
D
Re
lati
ve In
fect
ivit
y
(
% o
f n
o A
3)
0.0 0.2 0.4 0.6 0.810
100
Frequency of G -to-A
mutation (%)
hapI-GKE/SV-183
no A3
hapI-GKE/SV-200
hapII-RDD/SV-200
hapII-RDD/E56A
h
hapII-RDD/SV-183A
PO
BE
C3G
pT
R60
0
SV
-18
3
SV
-20
0
SV
-18
3
SV
-20
0
E5
6A
hapI-
GKE
hapII-
RDD
AP
OP
BE
C3G
pT
R60
0
SV
-18
3
SV
-20
0
SV
-18
3
SV
-20
0
E5
6A
hapI-
GKE
hapII-
RDD
00
6
8
10
12
6
8
10
12GG dinucleotide context GA dinucleotide context
2
4
2
4
Nu
mb
er
of
G-t
o-A
(pe
r si
ng
le c
lon
e)
pTR600
SV-1
83
SV-2
00
SV-1
83
SV-2
00
E56A
APOBEC3G
hapI-GKE hapII-RDD Controls
pTR600
SV-1
83
SV-2
00
SV-1
83
SV-2
00
E56A
APOBEC3G
hapI-GKE hapII-RDD Controls
ACCEPTED
on February 19, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from