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: viviana.simon@mssm.edu

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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-1s

    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.