Transcript
Page 1: 1 HUMAN APOBEC3H Ariana Harari*, Marcel Ooms*, Lubbertus

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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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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

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R D D

R D

DR

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L

LN

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Frequency in

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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

(%

) ACCEPTED

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Page 27: 1 HUMAN APOBEC3H Ariana Harari*, Marcel Ooms*, Lubbertus

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

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Page 28: 1 HUMAN APOBEC3H Ariana Harari*, Marcel Ooms*, Lubbertus

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

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Page 29: 1 HUMAN APOBEC3H Ariana Harari*, Marcel Ooms*, Lubbertus

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

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Page 30: 1 HUMAN APOBEC3H Ariana Harari*, Marcel Ooms*, Lubbertus

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

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Page 31: 1 HUMAN APOBEC3H Ariana Harari*, Marcel Ooms*, Lubbertus

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

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