THE INFLUENCE OF APOBEC3G AND DEOXYTHYMIDYLATE KINASE GENETIC DIVERSITY ON HIV-1 HYPERMUTATION AND RESPONSE TO TREATMENT
CRAIG STUART PACE, BSC (HONS)
The Centre for Clinical Immunology & Biomedical Statistics Royal Perth Hospital and Murdoch University Perth, Western Australia
This thesis is presented for the degree of Doctor of Philosophy of Murdoch University, 2006
I declare that this thesis is my own account of my research and contains as its main content work that has not previously been submitted for a degree at any tertiary education institution.
Craig Stuart Pace 15June2006
ABSTRACT
This thesis addresses two important topics in HIV-1 medicine; (i) the clinical relevance of pre-treatment G→A hypermutation and the contribution of host and viral genetics to its development and; (ii) the influence of genetic variation in host enzymes responsible for antiretroviral drug metabolism on response to therapy.
These themes are outlined below.
HIV-1 Hypermutation
At present, limited data exists regarding the relative roles of host encoded cytidine deaminases APOBEC3G and APOBEC3F in promoting G→A hypermutation of
HIV-1 proviral DNA in vivo , nor the clinical relevance of hypermutation or the influence of genetic diversity of the APOBEC3G locus and of the viral encoded vif protein that counteracts the action of APOBEC3G. The analyses contained within this thesis demonstrate that within the WA HIV cohort, clinically relevant hypermutation is restricted to a minority of individuals and is mediated predominantly by APOBEC3G. In this study, the presence of HIV-1 hypermutation had a substantially greater effect on plasma viremia than other known host antiviral factors such as CCR5 ∆32 or specific HLA-B alleles. Furthermore, the considerable genetic diversity of the vif gene is likely to make a greater contribution to the development of hypermutation than the limited genetic diversity of the APOBEC3G gene in Caucasians. These data indicate that G→A hypermutation is a clinically relevant phenomenon and may provide a fresh perspective to the area of HIV/AIDS therapies.
i Genetic Determinant of HIV-1 Treatment Response
Thymidine kinase 2 (TK2) and thymidylate kinase (dTMPK) are rate limiting
enzymes for the metabolism of the antiretrovirals d4T and AZT, respectively, and
are thus central to the antiviral efficacy and toxicity of these agents. However, the
genetic diversity of TK2 and dTMPK and their influence on toxicities associated
with their use is largely unknown. The results discussed in this thesis indicate that in
contrast to the highly conserved TK2 locus, the dTMPK locus of Caucasian
individuals, including regulatory regions potentially influencing transcription and
translation, is considerably polymorphic and organised into five common haplotypes.
The results regarding the contribution of dTMPK genetic variation to toxicities
associated with AZT therapy are encouraging. A common dTMPK haplotype had
significant, albeit modest, effect on haematological parameters (haemoglobin and
mean corpuscular volume) in HIV-infected patients, although no AZT-specific
treatment effect was observed in this relatively haematologically stable cohort. In
addition, another common dTMPK haplotype provided significant protection against
AZT-induced adipocyte mtDNA depletion in a pilot study of AZT- and d4T-treated
individuals. The dTMPK haplotypes characterised in this thesis should facilitate
further studies regarding dTMPK genetic variation in HIV-1 infection and response
to treatment, which are warranted from the clinical results presented herein.
ii TABLE OF CONTENTS
Abstract i Table of Contents iii List of Figures vii List of Tables xi Abbreviations xii Acknowledgements xv
CHAPTER ONE - POPULATION LEVEL ANALYSIS OF HYPERMUTATION AND ITS RELATIONSHIP WITH APOBEC3G AND VIF GENETIC VARIATION 1
1.1 GENERAL INTRODUCTION AND REVIEW OF THE LITERATURE 2
1.1.1 HYPERMUTATION 3 1.1.1.1 Description of HIV-1 Hypermutation 3 1.1.1.2 Cause of HIV-1 Hypermutation 3 1.1.1.3 Mechanism of APOBEC3-Mediated Hypermutation 5 1.1.2 APOLIPOPROTEIN B M RNA EDITING ENZYME CATALYTIC (-LIKE ) POLYPEPTIDES 7 1.1.2.1 Introduction to the AID/APOBEC Superfamily 7 1.1.2.2 Structure of AID/APOBEC Proteins 8 1.1.2.3 Evolution of the AID/APOBEC Superfamily 10 1.1.2.4 Expansion of the APOBEC3 Locus 11 1.1.2.5 Expression of APOBEC3 Proteins 16 1.1.2.6 Transcriptional Regulation of APOBEC3G 18 1.1.2.7 Post-Transcriptional Regulation of APOBEC3G 18 1.1.2.8 APOBEC3G-Vif Interaction 21 1.1.2.9 Virion Encapsidation of APOBEC3G 24 1.1.2.10 Antiviral Domains of APOBEC3G 26 1.1.3 VIRAL INFECTIVITY FACTOR (V IF ) 29 1.1.3.1 HIV Accessory/Regulatory Genes 29 1.1.3.2 Role of Vif 30 1.1.3.3 Vif -Mediated Degradation of APOBEC3G 32 1.1.3.4 Vif Encapsidation 35 1.1.3.5 Regulation of Vif 36 1.1.3.6 Vif-APOBEC3 Binding 37 1.1.4 CLINICAL SIGNIFICANCE 41 1.1.4.1 APOBEC3G Genetic Variation 41 1.1.4.2 Vif Genetic Variation 43 1.1.5 HYPOTHESIS & AIMS 45 1.1.5.1 Hypothesis 45 1.1.5.2 Aims 45
iii 1.2 MATERIALS & METHODS 46
1.2.1 PATIENT SELECTION 46 1.2.2 HIV-1 CLADE ASSIGNMENT 46 1.2.3 AMPLIFICATION AND SEQUENCING OF HIV-1 PROVIRAL DNA, MEASUREMENT OF PRE -TREATMENT HIV RNA LEVELS , HLA AND CCR5 GENOTYPING 47
1.2.4 ANALYSIS OF G→A SUBSTITUTIONS 48 1.2.5 ANALYSIS OF APOBEC3G AND APOBEC3F TARGET MOTIFS 49 1.2.6 ANALYSIS OF APOBEC3G- AND APOBEC3F-MEDIATED HYPERMUTATION 50 1.2.7 APOBEC3G ALLELE FREQUENCIES 51 1.2.8 STATISTICAL ANALYSIS 52
1.3 RESULTS 53
1.3.1 ANALYSIS OF G→A SUBSTITUTIONS 53 1.3.2 ANALYSIS OF APOBEC3G AND APOBEC3F DINUCLEOTIDE TARGET MOTIFS 54 1.3.3 CLASSIFICATION OF APOBEC3G- & APOBEC3F-HYPERMUTATED HIV-1 SEQUENCES 56
1.3.4 HIV-1 GENOMIC DISTRIBUTION OF G→A HYPERMUTATION 59
1.3.5 ASSOCIATION OF VIF AMINO ACID POLYMORPHISMS AND G→A HYPERMUTATION 59
1.3.6 APOBEC3G GENETIC VARIATION AND G→A HYPERMUTATION 65
1.3.7 G→A HYPERMUTATION AND HIV-1 VIREMIA 67
1.4 DISCUSSION 68
1.5 SUPPLEMENTARY DATA 73
CHAPTER TWO - CHARACTERISATION OF THE HUMAN DEOXYTHYMIDYLATE KINASE AND THYMIDINE KINASE 2 GENES AND THEIR INFLUENCE ON AZT AND D4T TOXICITIES 79
2.1 GENERAL INTRODUCTION AND REVIEW OF THE LITERATURE 80
2.1.1 AZT AND 4T THERAPY 80 2.1.1.1 Mode of Action of NRTIs 81 2.1.2 CLINICAL TOXICITIES OF THYMIDINE -ANALOGUE THERAPY 83 2.1.2.1 Haematological Toxicities 83
iv 2.1.2.2 Myopathy 85 2.1.2.3 Lactic Acidosis and Hyperlactataemia 86 2.1.2.4 Neuropathy 89 2.1.2.5 Lipoatrophy 90 2.1.2.6 Pancreatitis 91 2.1.3 MECHANISMS OF THYMIDINE -ANALOGUE INDUCED MITOCHONDRIAL DYSFUNCTION 93 2.1.3.1 Mitochondrial DNA Depletion 93 2.1.3.2 Non-Mitochondrial DNA Depletion Mechanisms of AZT Induced Mitochondrial Toxicity 96 2.1.4 NON -MITOCHONDRIAL DYSFUNCTION MECHANISMS OF AZT TOXICITY 97 2.1.4.1 Genotoxicity 97 2.1.4.2 Inhibition of Heme Synthesis 99 2.1.4.3 Nucleotide Pool Imbalance 100 2.1.5 PHOSPHORYLATION OF AZT & D 4T 101 2.1.5.1 Thymidine Kinase 2 102 2.1.5.2 Deoxythymidylate Kinase 104 2.1.5.3 Nucleoside Diphosphate Kinase 107 2.1.6 HYPOTHESES & AIMS 109 2.1.6.1 Hypothesis 109 2.1.6.2 Aims 109
2.2 MATERIALS & METHODS 110
2.2.1 IDENTIFICATION OF SNP S AND CHARACTERISATION OF HUMAN TK2 & DTMPK HAPLOTYPES 110 2.2.1.1 Amplification of dTMPK 111 2.2.1.2 Amplification of TK2 112 2.2.2 THE INFLUENCE OF D TMPK GENETIC VARIATION ON HAEMATOLOGICAL RESPONSE TO AZT THERAPY 114 2.2.3 THE INFLUENCE OF D TMPK GENETIC VARIATION ON AZT-INDUCED MT DNA DEPLETION 114 2.2.4 STATISTICAL ANALYSIS 117
2.3 RESULTS 118
2.3.1 IDENTIFICATION OF TK2 AND D TMPK SNP S 118 2.3.2 CHARACTERISATION OF D TMPK HAPLOTYPES 120 2.3.3 HAEMATOLOGICAL RESPONSE TO AZT AND D 4T 122 2.3.4 INFLUENCE OF D TMPK GENETIC VARIATION ON HAEMATOLOGICAL RESPONSE TO ZIDOVUDINE 124 2.3.4.1 Haemoglobin 124 2.3.4.2 Mean Corpuscular Volume 129 2.3.5 INFLUENCE OF D TMPK SNP S AND HAPLOTYPES ON AZT-INDUCED MT DNA DEPLETION 130
v 2.4 DISCUSSION 133
3 REFERENCES 138
vi LIST OF FIGURES
Figure 1.1 Replication of HIV-1 in non-permissive and permissive T 5 cells.
Figure 1.2 Schematic representation of the mechanism of 6 APOBEC3G mediated G →A hypermutation.
Figure 1.3 Domain organisation of AID/APOBEC proteins. 9
Figure 1.4 Structure of the cytidine deaminase catalytic domain. 10
Figure 1.5 APOBEC3 locus on chromosome 22. 12
Figure 1.6 Hypothetical scheme of the origin and divergence of the 13 APOBEC3 family in mammals.
Figure 1.7 Phylogeny of APOBEC3G in Primates. 15
Figure 1.8 APOBEC3G colocalises with mitochondria. 16
Figure 1.9 Northern blot analysis of APOBEC3 genes. 17
Figure 1.10 APOBEC3G mRNA levels are regulated by PKC and 19 MEK1/2.
Figure 1.11 Inducibility of HMM APOBEC3G complex formation in 20 primary CD4 + T cells with different stimuli, and correlation of the HMM complex with permissivity for HIV infection.
Figure 1.12 The APOBEC3G D128K mutation selectively allows 22 APOBEC3G to bind HIV-1 vif or SIVagm vif .
Figure 1.13 HIV-1 Vif is able to interact and form complexes with 23 APOBEC3G D128K mutant (A) but fails to reduce steady-state levels of the mutant APOBEC3G protein (B).
Figure 1.14 Mutations of the zinc coordinating (Cys97 andCys100, 25 1CCAA) and RNA binding (Phe70 and Tyr91, 1FYAA) residues of CD1 but not of the CD2 (2CCAA & 2FFAA) domain reduces HIV-1 encapsidation of APOBEC3G.
Figure 1.15 The Cytidine Deaminase Activity of APOBEC3G Is Not 27 Required for an Antiviral Effect.
vii Figure 1.16 Schematic representation of HIV-1 genome. 29
Figure 1.17 Replication of vif-positive and vif-negative HXB2viruses 31 in CD4+ human T-cell lines.
Figure 1.18 HIV-1 vif interacts with cellular proteins Cul5, ElonginB 33 and ElonginC (A) to promote the polyubiquitination and subsequent proteasomal-mediated degradation of vif - APOBEC3G complexes (B).
Figure 1.19 Vif C114S and C133S mutants are able to bind Cul5 (A) 35 but fail to ubiquitinate APOBEC3G (B) despite similar levels of vif , EloBC and Cul5 expression (C).
Figure 1.20 Phosphorylation of HIV-1 proteins by MAPK. 37
Figure 1.21 Stable coexpression of APOBEC3G and vif . 38
Figure 1.22 Vif peptides comprised of vif amino acids 85-99 and 169 40 to 192 significantly inhibit interaction between phage- displayed APOBEC3G and vif .
Figure 1.23 The APOBEC3G 186R variant associated with 42 accelerated progression to AIDS exhibits similar antiviral activity as the 186H variant.
Figure 1.24 G→A burden and G →A preference are highly correlated. 54
Figure 1.25 G→A hypermutation occurred predominantly in the 55 dinucleotide sequence contexts targeted by APOBEC3G (GG) and, to a lesser extent, APOBEC3F ( GA).
Figure 1.26 The consolidated 3G scores reflect a bimodal 57 distribution.
Figure 1.27 Non-specific G →A hypermutation (A), APOBEC3G- 60 specific G →A substitutions (B) and APOBEC3G- mediated hypermutation (C) scores are relatively constant along the HIV-1 genome.
Figure 1.28 Consensus vif amino acid sequence and polymorphism 62 frequency among non-hypermutated sequences.
Figure 1.29 Schematic representation of HM and truncated vif 64 proteins identified in vivo from putative translation of proviral DNA sequences.
viii Figure 1.30S Binding sites of oligonucleotide primers used for the 74 amplification (black) and sequencing (blue and red) of the entire human APOBEC3G gene, including 2kb upstream of the transcription binding site and 0.5kb 3’ of the 3’UTR.
Figure 1.31S Chromatograph results of sequencing pooled PCR. 76 products homozygous for alternative alleles at varying proportions to estimate the lower limit of allele detection by sequencing.
Figure 1.32S Power calculations used to determine sample size 78 required to detect significant differences in APOBEC3G allele frequencies between HM-3G and NH sequences based on the allele frequencies of the HM-3G sequences and those estimated from a pooled DNA control sample.
Figure 2.1 Structure of nucleoside reverse transcriptase inhibitors 82 and their corresponding deoxynucleoside.
Figure 2.2 Focal cytochrome oxidase deficiency is a unique and 86 consistent feature of AZT-associated myopathy.
Figure 2.3 Rise in population average venous lactate concentrations 88 after start of highly active antiretroviral therapy (HAART) in treated patients stavudine (dashed line) and zidovudine (solid line).
Figure 2.4 Proportion of patients with peripheral fat depletion 91 according to each body area, in the zidovudine and stavudine arms.
Figure 2.5 The effect of AZT on mtDNA/ nDNA ratio in vitro and 94 in vivo .
Figure 2.6 The effect of AZT on cell growth and haemoglobin 98 production.
Figure 2.7 TMPK is at the junction of the de novo and salvage 101 pathways of dTTP synthesis.
Figure 2.8 Comparison of phosphorylation of 2 µM D4T with 2 µM 102 AZT in CEM cells.
Figure 2.9 The relationship between MTT cytotoxicity (bars) and 105 AZT-MP and AZT-TP (trend lines) in five cell lines.
ix Figure 2.10 The reduced phosphorylation of nucleoside analogues is 107 due to the absence of the 3’-OH group which reduces the catalytic efficiency rather than the binding affinity.
Figure 2.11 Longitudinal mean corpuscular volume (MCV) profiles 114 of AZT-recipients
Figure 2.12 Longitudinal haemoglobin concentration profiles of 115 individuals initiating AZT therapy
Figure 2.13 Schematic representation of the human dTMPK gene and 117 location of identified SNPs.
Figure 2.14 Schematic representation of the human TK2 gene and 118 location of identified SNPs.
Figure 2.15 dTMPK haplotype pedigrees. 120
Figure 2.16 AZT recipients homozygous for 3’ haplotype B have 124 significantly higher on-treatment haemoglobin concentrations (A) and significantly less likely to have on-treatment haemoglobin concentrations less than 130 g/L (B).
Figure 2.17 No association existed between the 3’ haplotype B and 125 on-treatment haemoglobin concentrations in d4T- recipients.
Figure 2.18 Mean ∆ haemoglobin concentrations (A) and the 127 proportion of individuals that experienced and an increase in haemoglobin concentrations in response to AZT-therapy (B) were similar in individuals regardless of homozygosity for the 3’haplotype B or haplotype 3, respectively.
Figure 2.19 AZT recipients homozygous for the dTMPK haplotype 1 130 have significantly higher adipocyte mtDNA values than non-homozygous AZT recipients (A), despite similar duration of AZT exposure (B).
Figure 2.20 mtDNA values in AZT recipients homozygous for the 131 dTMPK haplotype 1 reflect that observed in recipients of non-thymidine-analogue therapy, HIV-1+ individuals not receiving antiretroviral therapy or HIV-1 seronegative controls.
x LIST OF TABLES
Table 1.1 Patient and sequence characteristics of patients harbouring 58 putative APOBEC3G-, APOBEC3F-hypermutated and non- hypermutated HIV-1 proviral DNA.
Table 1.2 APOBEC3G allele frequencies among 8 patients with marked 66 G→A hypermutation. Comparisons with a pooled DNA control sample and a published study.
Table 1.3S Oligonucleotide sequences used for the amplification and 73 sequencing of the entire human APOBEC3G gene, including 2.0 kb 5’ of the transcription start site and 1 kb 3’ of the transcription stop site.
Table 2.1 Oligonucleotide characteristics used for the amplification and 111 sequencing of the human dTMPK gene.
Table 2.2 Oligonucleotide characteristics used for the amplification and 112 sequencing of the human TK2 gene.
Table 2.3 Human deoxythymidylate kinase haplotypes identified from 121 studies of family members.
Table 2.4 Characteristics of AZT and d4T recipients. 122
xi ABBREVIATIONS
3TC lamivudine A adenine AACTG Adult AIDS Clinical Trials Group AAN antiretroviral-associated neuropathy ABC abacavir ADP adenosine diphosphate agm African green monkey AID activation-induced deaminase AIDS acquired immunodeficiency syndrome Ala alanine AMT 3’-amino-3’deoxythymdine ApoB apolipoprotein B APOBEC apolipoprotein B mRNA-editing enzyme Arg arginine Asn asparagine ATP adenosine triphosphate AZT zidovudine C cytosine CCR chemokine receptor CD cytidine deaminase CD4 cluster of differentiation maker 4 cDNA complimentary deoxyribose nucleic acid CFU-GM colony forming unit-granulocyte macrophage C-terminal carboxy-terminal Cul cullin Cys cysteine D aspartic acid d4T stavudine dA deoxyadenine dC deoxycytidine dCTP deoxycytidine triphosphate ddC zalcitabine ddI didanosine dG deoxyguanidine DNA deoxyribose nucleic acid dNTP deoxynucleoside triphosphate DP diphosphate dT deoxythymidine dTMPK deoxythymidylate kinase dTTP deoxythymidine triphosphate E glutamic acid Elo elongin ERK extracellular protein kinase F phenylalanine FDA Federal Drug Administration fl femtolitres FLT 3’-fluoro-3’-deoxythymidine
xii G guanidine Glu glutamic acid H histidine HAART highly active antiretroviral therapy HBV hepatitis B virus HCV hepatitis C virus HGH human growth hormone His histidine HIV human immunodeficiency virus HL hyperlactataemia HLA human leukocyte antigen HM-3F APOBEC3F-mediated hypermutation HM-3G APOBEC3G-mediated hypermutation HMM high molecular mass HSN HIV-associated sensory neuropathy H-strand heavy-strand HTLV human T cell leukemia virus I inosine IgC immunoglobulin C K lysine kDa kilodalton L leucine LA lactic acidosis LMM low molecular mass L-strand light-strand LTR long terminal repeat Lys Lysine Mac macaque MAPK mitogen activated protein kinase MCV mean corpuscular volume MEK mitogen-activated protein kinase 1 and 2 MP monophosphate mRNA messenger ribonucleic acid mtDNA mitochondrial deoxyribose nucleic acid MuLV murine leukemia virus n number Nc nucleocapsid NCBI National Centre for Biotechnology Information NCI National Cancer Institute NDK nucleoside diphosphate kinase nDNA nuclear deoxyribose nucleic acid Nef negative factor NH non-hypermutated NNRTI non-nucleoside reverse transcriptase inhibitors NRTI nucleoside reverse transcriptase inhibitors N-terminal amino-terminal P proline PBMC peripheral blood mononuclear cells PCR polymerase chain reaction PHA phytohemagglutinin
xiii Phe phenylalanine Pi inorganic phosphate Pro proline Q glutamine R arginine Rev regulator of viral protein synthesis RNA ribonucleic acid RT reverse transcriptase S serine SD standard deviation Ser serine SIV simian immunodeficiency virus SNP single nucleotide polymorphism SOCS suppressor of cytokine signalling T thymidine Tat transactivating transcription factor TDF tenofovir Thr threonine TK thymidine kinase TNF tumour necrosis factor TP triphosphate TSS transcription start site Tyr tyrosine U uridine UTR untranslated region Vif viral infectivity factor Vpr viral protein R Vpu viral protein U wt wild type Y tyrosine
xiv ACKNOWLEDGEMENTS
Firstly, I would like to thank Professors Simon Mallal and Ian James for giving me the opportunity to study for a PhD degree. Without their support, funding and most of all faith in me to complete, firstly an Honours degree, and then a PhD degree, I would not be in this position today. In that regard, I would also like to thank Neill
Hodgen, who was instrumental in me obtaining a position with the Centre when I first finished my undergraduate studies. I would also like to thank my supervisor, Dr
David Nolan, for his encouragement, advice and invaluable time; no matter how busy he was, he was always able to make the time to discuss any aspect of my thesis.
My gratitude also extends to Jean Keller for performing the database queries required for the hypermutation analyses and statistical advice, Dr Silvana Gaudieri for general methodology issues, Dr Larry Park for HIV-1 clade assignment, Dr
Corey Moore for establishing the HIV-1 sequence database and Filipa Carvalho for performing a large proportion of the HIV-1 sequencing.
I would also like to thank my fellow S blockers; PhD students Nicole Prada, Coral-
Ann Almeida and Bree Foley; computer staff Mark Shaw and Iain Bradley and
Niamh Keene for thesis advice. Finally, I’d like to thank my family for their encouragement.
xv
1
CHAPTER ONE
1 POPULATION LEVEL ANALYSIS OF HYPERMUTATION AND
ITS RELATIONSHIP WITH APOBEC3G AND VIF GENETIC
VARIATION 1.1 GENERAL INTRODUCTION AND REVIEW OF THE LITERATURE
Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G
(APOBEC3G) and the closely related APOBEC3F are recently identified anthropoid-specific proteins that restrict human immunodeficiency virus-1 (HIV-1) replication by deaminating cytosine residues in intermediary single-stranded HIV
DNA. This host antiviral strategy thereby introduces DNA editing errors into the retroviral genome sequence, resulting in the fixation of an inordinate number of proviral HIV DNA guanine to adenine (G →A) substitutions referred to as
hypermutation. Previous studies have defined polynucleotide motifs within single-
stranded DNA that are preferentially targeted by APOBEC3G (resulting in proviral
DNA GG→AG substitutions) and APOBEC3F ( GA→AA). These studies have also identified HIV-1 viral infectivity protein ( vif ) as the principal viral factor that counteracts APOBEC3-mediated DNA editing by promoting the degradation of
APOBEC3-vif complexes via the proteasomal pathway. Recent in vitro data also indicate that APOBEC3G and APOBEC3F are partially resistant to vif , suggesting a more fundamental role for these APOBEC3 proteins in directing HIV-1 genetic variation.
However, at present little is known of the disease-modulating effects of APOBEC3G and/or APOBEC3F in HIV-infected patients, and it is uncertain if APOBEC3- mediated HIV DNA editing in vivo requires permissive conditions such as defective vif activity and/or APOBEC3 genetic variation. This chapter of the thesis, accepted for publication in Journal of Virology (Pace et al , 2006), aims to address these issues at the population level and begins with an extensive review of the relevant literature.
2 1.1.1 HYPERMUTATION
1.1.1.1 DESCRIPTION OF HIV-1 HYPERMUTATION
Hypermutation, defined as extensive, monotonous guanine to adenine (G→A) substitutions, was first described by Vartanian and colleagues 1 and Li and colleagues 2. In 1991, Vartanian et al ,1 described seven clones of HIV-1 proviral
DNA in which over 90% of all mutations were G →A and Li et al ,2 reported quasispecies with 8.2% nucleotide differences comprised of almost exclusively
G→A substitutions from uncultured brain tissue from a patient with AIDS dementia complex. In both studies, 63-90% of the G→A substitutions affected guanidine residues within GA dinucleotides. In contrast, Fitzgibbon and colleagues 3 described a second type of G→A hypermutation from longitudinal PBMC proviral DNA from an AIDS patient that preferentially affected one or more 5’ guanidines in runs of guanidines. Adenine to inosine (A-I) hypermutation which has been described in measles and vesicular stomatitis genomes has not been observed from HIV-1 genomes.
1.1.1.2 CAUSE OF HIV-1 HYPERMUTATION
Initial describers of G→A hypermutation attributed the phenomena to base mispriming, temporary dislocation of the primer and template and the low fidelity of
HIV-1 reverse transcriptase (RT) and/or altered intracellular nucleotide pool balances 4-7.
3 Work from Martinez and colleagues 6 demonstrated that G→A hypermutation could
be achieved in vitro using HIV-1 RT and biased deoxynucleotide triphosphate
(dNTP) concentrations, in particular, low ratio of deoxycytidine triphosphate
(dCTP): deoxythymidine triphosphate (dTTP), in a dose-dependent manner. HIV-1
RT is particularly sensitive to such biased deoxynucleotide pools, generating
significantly more G→A changes than RT of avian myeloblastosis virus or Moloney
murine leukemia virus in a suboptimal dCTP environment 5. However, suboptimal
dCTP concentrations are unlikely to produce G→A hypermutation specifically in the
GA and GG contexts. Vartanian et al 7 agreed that suboptimal dCTP concentrations
would account for type 2 G→A hypermutation (one or more 5’ guanidines in runs of
guanidines) due to the progressive depletion of dCTP in the microenvironment
caused by runs of guanidines, but suggested that dislocation mutagenesis consequent
to dCTP depletion was the mechanism for hypermutation in the GA context.
The theory that hypermutation was caused by biased deoxynucleotide pools appeared less likely following the discovery that hypermutation occurred only in cells and cell lines that were non-permissive for HIV-1 infection, despite having similar deoxynucleotide pools to permissive cells 8. Non-permissive cells are cells in which
the viral-encoded viral infectivity factor ( vif ) is indispensable for HIV-1 infection
(PBMCs, HUT78, H9, CEM) as opposed to permissive cells in which vif is
dispensable for HIV-1 infectivity (Figure 1.1).
Using cell-fusion experiments, Simon and colleagues9 and Madani & Kabat 10 discovered that the non-permissive phenotype was dominant, suggesting that non- permissive cells produce an antiviral factor that is suppressed by vif , and through
4 cDNA subtraction studies, Sheehy and colleagues 11 discovered that APOBEC3G was abundantly expressed in non-permissive cells but only minimally expressed, or absent, in a variety of lymphoid and non-lymphoid permissive cells. Subsequently,
APOBEC3G 12-14 , APOBEC3F 15-17 and APOBEC3B 18 were found to induce G→A hypermutation in HIV-1 proviral DNA in vitro . Based on the specific sequence contexts that APOBEC3 proteins exhibit in regard to cytidine deamination sites and their tissue distribution (section 1.1.2.5), APOBEC3G 19,20 and APOBEC3F 15 appear to mediate HIV-1 hypermutation in vivo .
Figure 1.1 Replication of HIV-1 in non-permissive and permissive T cells. CEM and CEM-SS cells were challenged with normalized stocks of wild-type or ∆vif X4-tropic viruses, and virus replication was monitored as the supernatant accumulation of p24 gag . Taken from reference 11 (Reprinted by permission from Macmillan Publishers, Ltd) [Nature , 2002;418:646-50].
1.1.1.3 MECHANISM OF APOBEC3-MEDIATED HYPERMUTATION
As illustrated in Figure 1.2, the HIV-1 RNA genome is reverse transcribed in the cytoplasm by the viral-encoded reverse transcriptase producing the first (negative)
DNA strand. Following RNase H-dependent dissociation of the RNA-DNA hybrid,
APOBEC3 proteins deaminate the cytidine molecules on the negative strand cDNA,
5 due to their single-strand DNA specificity 21 , yielding uracil molecules. During replication of the negative strand cDNA, the uracil molecules are identified by the
DNA polymerase as thymidine molecules and therefore result in the incorporation of adenine molecules in place of the guanidine molecules in the proviral DNA precursor, or G→A hypermutation. Alternatively, uridine containing cDNAs are
targeted by cellular uracil DNA glycosylase, which excises the contaminating
uridine molecule, leaving an abasic site which is endonucleolytically cleaved by
apyrimidinic endonucleases yielding severed proviral DNA precursors that are
incapable of being integrated into the cellular genomic DNA 14 .
Figure 1.2 Schematic representation of the mechanism of APOBEC3G mediated G →→→A hypermutation. Viral RNA is reverse transcribed by reverse transcriptase to generate the first (negative) strand DNA, which upon RNase H-mediated degradation of the genomic RNA, becomes single-stranded and targeted by APOBEC3G. The deamination of cytosine by APOBEC3G yields uridine molecules, which are either identified as thymidine molecules by DNA polymerase and result in the incorporation of adenine molecules, and once integrated, become fixated as G →A substitutions in proviral DNA. Alternatively, uracil-containing DNA is targeted by uracil DNA glycosylase, which excises the contaminating uracil, leaving an abasic site which is endonucleolytically cleaved by apyrimidinic endonucleases yielding severed proviral DNA precursors. Taken from reference 14 (reprinted by permission from Elsevier Science) [ Cell , 2003;113:803-9].
6 1.1.2 APOLIPOPROTEIN B M RNA EDITING ENZYME CATALYTIC (-
LIKE ) POLYPEPTIDES
1.1.2.1 INTRODUCTION TO THE AID/APOBEC SUPERFAMILY
The AID/APOBEC proteins are a superfamily of DNA and RNA cytidine deaminases. The superfamily can be segregated into activation-induced deaminase
(AID), apo lipoprotein-B RNA editing enzyme, catalytic polypeptide 1 (APOBEC1),
APOBEC2 and APOBEC3 subgroups based on the sequences surrounding the conserved zinc-binding motifs characteristic of all cytidine deaminases 22 .
The founding member, APOBEC1, an RNA deaminase is expressed exclusively in the small intestine 23 . APOBEC1 is the catalytic component of a complex that deaminates cytidine 6666 of the apolipoprotein B mRNA polynucleotide, creating a premature stop codon that results in the tissue-specific production of a truncated form of apoB-100, termed apoB-48 24 .
AID is a single stranded DNA deaminase expressed exclusively in activated B lymphocytes 25 and functions in adaptive humoral immune response by potentiating antibody gene diversification through somatic hypermutation and gene conversion of the immunoglobulin V gene 26 and switch recombination of the IgC gene 27,28 .
APOBEC2 is expressed exclusively in heart and skeletal muscle but its function is to date unknown 29 . Mice deficient in APOBEC2 through gene targeting reveal
APOBEC2 is unessential for mouse development, survival or fertility 30 .
7 APOBEC3 family members are single stranded DNA cytidine deaminases, some of which provide innate antiviral restriction of retroviral (APOBEC3G) 13,31 ,
hepadnaviral (hepatitis B) (APOBEC3B, -3C, -3F and -3G) and endogenous
retroelement replication intermediaries 21,32 by cytidine deamination and non-cytidine
deamination mechanisms. Some APOBEC3 family members are abundantly
expressed in a variety of tumour cell lines suggesting they may also have a role in
neoplastic transformation 33 .
Recently, a new subfamily of the AID/APOBEC superfamily, termed APOBEC4, has been identified from iterative databases with readily identifiable orthologs in mammals, chicken, and frog, but not fishes. In mammals, APOBEC4 is expressed primarily in testis which suggests the possibility that it is an editing enzyme for mRNAs involved in spermatogenesis 34 .
1.1.2.2 STRUCTURE OF AID/APOBEC PROTEINS
By analogy to APOBEC1, the functional domains of APOBEC3G can be separated into an N-terminal domain, a first active site, a first linker region, a pseudoactive site, a second active site, a second linker region, and a C-terminal domain 33 (Figure
1.3). All APOBEC proteins contain one or two copies of a His/Cys-X-Glu-X23-28 -
Pro-Cys-X2-Cys signature motif that is characteristic of all cytidine deaminases and represents the active site 35 . This motif contains zinc-binding ligands and a glutamate involved in proton shuffling.
8
Figure 1.3 Domain organisation of AID/APOBEC proteins. The proteins were aligned on the active site residues of APOBEC1 and the predicted active site residues at the N terminus of the APOBEC3 proteins. Taken from reference 33 (reprinted by permission from Elsevier Science) [Genomics , 2002;79:285-296].
The secondary structure of any APOBEC protein is yet to be determined; however computer-assisted secondary structure prediction programs predict the secondary structure of the catalytic core is well conserved between nucleotide and polynucleotide cytidine deaminases 35 . Crystallographic studies of nucleotide cytidine deaminases from Escherichia coli 36 , Bacillus subtilus 37 , Saccharomyces cerevisiae 38
39 α α α and humans reveal a core β1β2 1β3 2β4β5 arrangement. Two -helices, that contain the His, Cys and Glu residues that are required for zinc- coordination, proton transfer and catalysis are supported by the five β-sheets in a parallel position 40 (Figure 1.4A).
The most striking difference between the predicted secondary structures of APOBEC proteins and the established structures of nucleotide cytidine deaminases is that all
APOBEC proteins have an intervening α-helix ( α3) between β3 and β4, which is lacking in nucleotide cytidine deaminases (Figure 1.4B). Interestingly, the α3-helix contains a critical amino acid residue required for species-specific interactions with
9 HIV-1 and SIV vif proteins. Therefore, APOBEC proteins are predicted to conform
α α α α α to a β1β2 1β3 2β4 3β5 arrangement rather than the β1β2 1β3 2β4β5 arrangement characteristic of nucleotide cytidine deaminases 40 .
Figure 1.4 Structure of the cytidine deaminase catalytic domain. (A) A mixed β-sheet is formed α α α by the consensus β1β2 1β3 2β4β5 motif, which supports the proper positioning of the -helices that contain the catalytic and zinc-coordinating residues. (B) Between β4 and β5, all APOBEC proteins α α contain a predicted -helical structure 3, which in APOBEC3G, is adjacent to residue 128 and is associated with the species-specific interaction with vif . Taken from reference 35 (reprinted by permission from Elsevier Science) [Virology , 2005;334:147-53].
1.1.2.3 EVOLUTION OF THE AID/APOBEC SUPERFAMILY
Phylogenetic analysis of AID/APOBEC sequences suggest that APOBEC1 and
APOBEC3 are most likely to have arisen by duplication of the AID locus. Consistent
with gene duplication, the AID, APOBEC3 and APOBEC1 genes have similar
location of intron/exon boundaries 22 . Analysis of the amino acid sequences of the
APOBEC active sites, linker and repeated linker sequences suggest that APOBEC1 is more closely related to AID, and then to APOBEC2, in both the active site
(consisting of three zinc ligands, critical residues for substrate binding, and a glutamate involved in proton shuffling during catalysis) and linker sequences, but all
10 three are more distant from APOBEC3 than from each other 33 . AID and APOBEC2 homologues can be traced back to cartilaginous fish and bony fish, respectively, with neither protein identifiable among nonchordates, nor the invertebrate chordate,
Ciona intestinalis 22 . APOBEC1 and APOBEC3 orthologs are restricted to mammals.
However, unlike the single APOBEC3 gene in mouse and rat, there are 8 APOBEC3 genes in primates and humans, designated APOBEC3A to APOBEC3H, arranged in tandem and each orientated with a centromeric 5’ end on chromosome 22q12- q13.2 33 . Synteny between the human, chicken and pufferfish genomes is evident in the region flanking the APOBEC3 locus and thus, while the APOBEC3 genes themselves are missing, the genes flanking the APOBEC3 locus are conserved in chicken and pufferfish, supporting the later origin of the APOBEC3 locus. All
APOBEC3 orthologs are present in the chimpanzee genome and therefore it appears that the expansion of this locus occurred at the beginning of the primate evolution 22 .
1.1.2.4 EXPANSION OF THE APOBEC3 LOCUS
In contrast to mice and other non-primate mammals, which have only a single
APOBEC3 gene, the APOBEC3 locus of primates and humans has undergone expansion and tandem duplication, resulting in the acquisition of seven APOBEC3 genes (APOBEC3A-3D and APOBEC3F-H) and one pseudogene (APOBEC3E).
The APOBEC3B and APOBEC3G genes each comprise eight exons, APOBEC3F comprises seven exons, APOBEC3A comprises five exons, APOBEC3C and
APOBEC3E each comprise four exons and APOBEC3D comprises three exons
(Figure 1.5). APOBEC3B, APOBEC3G and APOBEC3F share identical intron/exon
11 boundaries, except APOBEC3F terminates after exon 7. In APOBEC3B,
APOBEC3G and APOBEC3F, exons 2, 3 and 4 are duplicated in exons 5, 6 and 7, with introns 1-4 in the same position as introns 5-7. Between exons 4 and 5 of
APOBEC3B and APOBEC3F, a duplicated exon one-like sequence containing the initiating methionine and the other five amino acids of exon 1, is skipped rather than spliced due to a thymidine to guanidine substitution within a 3’-splice donor site at position 2 of the intron. No such exon skipping is present in APOBEC3G 33 . The
sequence corresponding to the third intron of the APOBEC3 genes is not spliced out
of the APOBEC3D mRNA sequence and has a continuous open reading frame with
the equivalent exons three and four to the other APOBEC3 genes. This however, is
not due to defective 3’ splice donor and acceptor sites 33 .
Figure 1.5 APOBEC3 locus on chromosome 22. The exons are numbered and boxed and assembled into cDNA where they are known to be expressed. The numbers corresponding to The Sanger Centre chromosome 22 sequence (bK150C2) are shown. Asterisks (*) indicate APOBEC3A exon one-like sequence present in APOBEC3B and 3F but skipped. Taken from reference 33 (reprinted by permission from Elsevier Science) [ Genomics , 2002;79:285-296].
12 The zinc-coordinating domains of APOBEC3 proteins provide evidence of their evolution. The zinc-coordinating domains can be grouped into two major clades, labelled Z1 and Z2. The Z1 domains, common to other AID/APOBEC family members, retain the SWS motif upstream of the PC-C motif unlike Z2 domains which contain a threonine residue in place of the first serine residue; Z1 domains are further divisible into Z1a and Z1b. As represented in Figure 1.6, the double domained rodent APOBEC3 is a Z1-Z2 composite, while all human APOBEC3’s are either single or double Z1 domained, except for APOBEC3H which consists of a single Z2 domain. It is therefore likely that the APOBEC3 locus evolved from a common ancestor that harboured both Z1 and Z2 domains either in the single- or double-domained APOBEC3 proteins 22 .
Figure 1.6 Hypothetical scheme of the origin and divergence of the APOBEC3 family in mammals. Two ancestral domains, Z1 domains (circles) and Z2 domains (squares) either constituted a double-domained APOBEC3 or two single domained APOBEC3s. The Z1 domains in primates is then considered to have diversified into Z1a (open circles) and Z1b (closed circles) subtypes. Taken from reference 22 (reprinted by permission from Oxford University Press, Ltd) [ Mol Biol Evol , 2005;22:367-77].
APOBEC3B, APOBEC3G and APOBEC3F are unique among the APOBEC proteins by containing a duplicated active site, insert and linker peptide and part of
13 the pseudoactive site. It has been proposed therefore, together with phylogenetic analyses, that duplication of either APOBEC3A, APOBEC3C, APOBEC3D or
APOBEC3E led to the origin of either APOBEC3B or APOBEC3G, and subsequently duplication of APOBEC3B or APOBEC3G formed APOBEC3F 33 . The
similarity in intron 4 of APOBEC3B and APOBEC3F is evidence of the former
while the similarity in the promoter sequence of APOBEC3D, APOBEC3F and
APOBEC3G is evidence of the latter 33 .
Analysis of the APOBEC3 genes from ten primate species representing 33 million
years of evolution indicates that APOBEC3G (Figure 1.7), APOBEC3B,
APOBEC3D and APOBEC3C and amino acids 117-250 of APOBEC3F, has been
subject to positive selection throughout the history of primate evolution, evident
from the excess of non-synonymous substitutions relative to synonymous
substitutions ( ω)41 . Interestingly, despite the lack of evidence that APOBEC3E is a functional gene, it exhibited the highest ω value seen among any human-chimp
comparison leading the authors to argue that it is an active participant in some form
of genetic conflict 41 . APOBEC3A was the only APOBEC3 gene that exhibited
evidence of purifying selection (referring to an excess of synonymous substitutions
relative to non-synonymous substitutions due to non-synonymous substitutions being
more likely to be deleterious and therefore typically culled out by selection).
Primate lentiviruses are no older than 1 million years and therefore have not been in
the primate population long enough to account for the positive selection of the
mentioned APOBEC3 proteins. The authors conclude that the ancient, constant
pressure of positive selection on APOBEC3 proteins, in particular APOBEC3G, in
14 primates suggest that at least some of its evolution may be due to genetic conflict in the germline, where APOBEC3G is abundantly expressed and where genome restricted mobile genetic elements need to transpose to ensure survival, rather than in the lymphocytes. They argue that over time, the steady retrotransposition of endogenous retroviruses that have recently been shown to be targets of APOBEC3 proteins 42-44 are likely to be more detrimental to a species than scattered, episodic interactions with viruses.
Figure 1.7 Phylogeny of APOBEC3G in Primates. APOBEC3G has been under positive selection for at least 33 million years. Numbers represent ω (non-synonymous: synonymous ratio; actual numbers in parentheses). HIV/SIV-infected species are indicated by asterisks (*). Taken from reference 41 (No permission required to reprint).
Alternatively, I identified a putative mitochondrial localisation signal in the amino acid sequence of APOBEC3G, APOBEC3F and APOBEC3H and it was suggested that mitochondrial localised APOBEC3 proteins may have contributed to the establishment or maintenance of the asymmetrical nucleotide distribution
15 characteristic of mammalian mitochondrial DNA (G-rich H-strand and C-rich L- strand) (David Nolan, pers comm ). Utilising confocal microscopy and western blot
analyses, endogenous and recombinant APOBEC3G and -3F proteins were indeed
found to localise to mitochondria and analyses of the dinucleotide composition and
codon usage of the human mitochondrial DNA Cambridge reference sequence seem
to support the hypothesis (Figure 1.8, Prada et al , in preparation ).
A B MitoTraker Protein Merged primary Ab tot mt c
anti APOBEC3G PBMC APOBEC3G-HA serum
GAPDH PBMC anti APOBEC3G serum COX IV
anti APOBEC3G PBMC serum
PBMC anti APOBEC3G serum
3G-293T anti APOBEC3G serum
3G-293T murine anti-HA mAb
Figure 1.8 APOBEC3G colocalises with mitochondria. (A) Endogenous (PBMC) and recombinant (3G-HEKT cells) APOBEC3G proteins were found to colocalise with mitochondria by confocal microscopy and (B) recombinant APOBEC3G by western blot analyses; red staining represents mitochondria; green staining represents APOBEC3G protein; yellow staining represents co- localisation (Prada et al , in preparation).
1.1.2.5 EXPRESSION OF APOBEC3 PROTEINS
All members of the AID/APOBEC superfamily exhibit tissue-specific expression.
As mentioned in 1.1.1.4, AID is specifically expressed in activated B lymphocytes 23 ,
APOBEC1 in intestinal epithelial cells 25 , APOBEC2 in skeletal and heart muscle 29
16 and APOBEC4 primarily in testis 34 . Similarly, APOBEC3 proteins are expressed in a tissue-specific manner. APOBEC3G, APOBEC3F and APOBEC3C are expressed in spleen, peripheral blood lymphocytes, ovary and testes (Figure 1.9). APOBEC3B,
APOBEC3C, and to a lesser extent, APOBEC3A, are abundantly expressed in a variety of tumour cells including colorectal adenocarcinoma cells, promyelocytic leukemia cells and lung carcinoma cells 33 .
Figure 1.9 Northern blot analysis of APOBEC3 genes. Multiple tissue and multiple tumour cell line northern blots were analysed for sites of expression of APOBEC3B , 3G , and 3C . Site of expression of actin control is also shown. Taken from reference 33 (reprinted by permission from Elsevier Science) [Genomics , 2002;79:285-96].
17 1.1.2.6 TRANSCRIPTIONAL REGULATION OF APOBEC3G
Unlike many other antiviral factors, such as adenosine deaminases, ribonuclease L and Mx GTPase, transcription of APOBEC3G mRNA is unaffected by type 1 or type
2 interferons or tumour necrosis factor-α (TNF α)45 or by HIV-1 infection per se 46,47 .
Data from Rose and colleagues 45 presented in Figure 1.10 suggest that transcription
of APOBEC3G mRNA is regulated by the cell cycle via a protein kinase cascade
involving protein kinase C-α and -β1, mitogen-activate protein kinase 1 and 2
(MEK) and extracellular signal-regulated protein kinase (ERK) 1 and 2. This is
consistent with data from Stopak and colleagues 46 that indicate an increase in
APOBEC3G in resting T lymphocytes following activation with interleukin-2 and
phytohemagglutinin. In addition, the 5’-untranslated region of APOBEC3G contains
potential binding sites for transcription factors Ets-1, c-Myc and Elk-1, each of
which are activated by the mitogen-activated protein kinase pathway 45,48 .
1.1.2.7 POST -TRANSCRIPTIONAL REGULATION OF APOBEC3G
Similar to the transcriptional regulation of APOBEC3G, the post-transcriptional regulation of APOBEC3G is also achieved by the cell cycle via the assembling of
APOBEC3G into an inactive high molecular mass (HMM) complex. Work by Chiu and colleagues 31 indicates that in unstimulated peripheral blood CD4 + T cells and monocytes, where HIV-1 replication is typically blocked, APOBEC3G resides almost exclusively as a low molecular mass (LMM) of approximately 46-kDa.
However, mitogen activation of CD4 + T cells and differentiation of monocytes to
macrophages induces the recruitment of the endogenous 46-kDa LMM APOBEC3G
18
Figure 1.10 APOBEC3G mRNA levels are regulated by PKC and MEK1/2. A, H9 cells were treated with the general PKC inhibitors, PKC inhibitor 20-28 (100 nM) or Calphostin C (50 nM), or with the PKC-specific inhibitors, Go6976 (200 nM) and Ro-32-0432 (100 nM), for 1 h prior to the addition of PMA (100 nM). B, H9 cells were treated with the specific MEK inhibitors U0126 (20 µM) or PD98059 (50 µM) or mock-treated with Me 2SO alone for 1 h prior to the addition of PMA (100 nM) or 4 α-PMA (100 nM), an inactive phorbol ester. The Me 2SO concentrations were equal in all of the cultures. RNA was isolated from cells at the indicated times and was analysed by quantitative real-time PCR for APOBEC3G mRNA levels. C, Northern blot analysis of H9 cells treated with U0126 (20 µM) or mock-treated for 1 h prior to the addition of PMA (100 nM) using a [ 32 P]-labelled DNA probe specific for APOBEC3G. The Northern blot was subsequently probed with a [ 32 P]- labelled DNA probe for the S2 ribosomal RNA loading control. Taken from reference 45 (No permission required to reprint).
19 protein into a HMM ribonucleoprotein complex, of approximately 700-kDa in mass, and correlated with a sharp increase in permissiveness for HIV infection (Figure
1.11). Specifically, they noted that progression into the G1b, but not the S phase of the cell cycle was necessary for HMM APOBEC3G complex formation.
Figure 1.11 Inducibility of HMM APOBEC3G complex formation in primary CD4 + T cells with different stimuli, and correlation of the HMM complex with permissivity for HIV infection. (A) APOBEC3G complexes were identified by FPLC, SDS–PAGE and immunoblotting with anti- APOBEC3G antibody. Single-round infection experiments were performed with the VSV-G- pseudotyped NL4-3 HSA virus. Ranges of HIV infectivity were determined by normalizing the percentage of HSA-positive cells obtained with each treatment to the response in cells stimulated with PHA and IL-2 (assigned a value of 100%). HIV infectivity: >70% (high, +++); 30–70% (moderate, ++); 5–30% (low, +); <5% (none, -). Scoring was based on the infectivity data obtained from cells isolated from four healthy donors. PMA, phorbol 12-myristate 13-acetate. Taken from reference 31 (reprinted by permission from Macmillan Publishers, Ltd) [ Nature , 2005;435:108-14].
20 1.1.2.8 APOBEC3G-VIF INTERACTION
Lentiviral vif proteins generally exhibit high species-specificity such that it has been hypothesised that vif may be a key regulator of the primate species tropism of primate immunodeficiency viruses 9,49 . The chimpanzee variant of SIV, which gave rise to HIV-1, and sooty mangabey SIV, which gave rise to HIV-2, encode vif proteins that are able to block human APOBEC3G, thus allowing their crossover into human populations as zoonotic infections. In contrast, the vif protein encoded by
African green monkey (agm) SIV is active against the agm APOBEC3G protein but does not inhibit human APOBEC3G and thus replicates poorly in primary human cells 9,49 .
Despite the high degree of homology between human and agm APOBEC3G (77%), several laboratories have demonstrated that the species-specific interaction of
APOBEC3G and vif is highly dependent on a single APOBEC3G amino acid 50-53 .
These in vitro studies demonstrated that mutation of the aspartic acid residue at position 128 of human APOBEC3G to that observed in agm (lysine) renders the mutant human APOBEC3G resistant to HIV-1 vif and sensitive to SIVagm vif and conversely, agm APOBEC3G that contained the reciprocal K128D mutation became resistant to SIVagm vif and sensitive to HIV-1 vif (Figure 1.12A). Furthermore, the exchange at position 128 switched the specificity with which APOBEC3G bound
SIVagm or HIV-1 vif (Figure 1.12B). This species-specificity suggests APOBEC3G and vif interact directly rather than through an RNA or protein intermediary 51 .
21 A
B
Figure 1.12 The APOBEC3G D128K mutation selectively allows APOBEC3G to bind HIV-1 vif or SIVagm vif . (A) 293T cells were cotransfected with the pNL-Luc-HXB_ Vif proviral indicator construct together with the HIV-1 Vif expression plasmid pg Vif or the SIVagm Vif expression pSIVagm Vif , or a control plasmid, and finally with plasmids expressing wild-type or mutant forms of h3G or agm3G. At 44 h after transfection, the supernatant media were harvested and used to infect CD4 +, CXCR4 + 293T cells. A further 28 h later, the infected cells were lysed and the level of virus- encoded Luc activity was quantified. Data are expressed relative to the level of Luc activity induced by virus derived from a culture transfected with pNL-Luc-HXB_ Vif in the absence of any APOBEC3G or Vif expression plasmid, which was arbitrarily set at 100. The average of three independent experiments with SD is indicated. (B) Transfected 293T cells with the HIV-1 Vif expression plasmid pg Vif and a plasmid encoding the indicated HA-tagged APOBEC3G protein. At 48 h after transfection, the cells were lysed and subjected to immunoprecipitation by using a mouse anti-HA mAb. Proteins present in the total lysate (T) or immunoprecipitate (IP) were then separated by gel electrophoresis, transferred to a nitrocellulose filter, and analysed by Western blot analysis by using rabbit polyclonal antisera specific for the HA tag ( Upper ) or the HIV-1 Vif protein ( Lower ). The negative (Neg) control lane was transfected with the control pcDNA3 plasmid. This experiment used 2% of the total lysate and 10% of the immunoprecipitate in each lane. Taken from reference 53 (No permission required to reprint).
However, it has been reported that a fragment of APOBEC3G consisting of residues
54-124 is able to specifically interact with HIV-1 in transfected cells 22 . Thus, it is
likely that position 128 does not directly participate in vif -APOBEC3G binding, but
22 rather facilitates complex formation 53 . Consistent with such a conclusion, Xu and colleagues 54 also observed inhibited HIV-1 replication in the presence of the D128K
APOBEC3G mutant and wild type vif however, in contrast to previous studies, the
APOBEC3G D128K mutant was able to form complexes with HIV-1 vif (Figure
1.13A) but steady-state levels of the D128K APOBEC3G mutant were not depleted and failed to be degraded (Figure 1.13B). This was not due to a disruption of a structural element that serves as a recognition signal for proteasomal degradation because the D128K mutant was efficiently degraded when complexed with
SIVmac239 vif or HIV-2 vif . Taken together, these results suggest that the HIV-1 vif -
APOBEC3G interaction induces a conformational change in APOBEC3G that exposes the asp-128 residue which synergistically binds vif , promoting APOBEC3G inhibition 50 and initiates vif -APOBEC3G complex degradation 54 .
A B
Figure 1.13 HIV-1 Vif is able to interact and form complexes with APOBEC3G D128K mutant (A) but fails to reduce steady-state levels of the mutant APOBEC3G protein (B). (A) Wild-type and Apo12 (contains D128K mutation) APOBEC3G interact with HIV-1 Vif as determined by coimmunoprecipitation. An anti-myc antibody attached to magnetic beads was used to immunoprecipitate the wild-type APOBEC3G and the Apo12 mutant proteins from cell lysates. The cellular proteins that were coimmunoprecipitated were analysed for the presence of Vif by using an anti-Vif antibody. (B) Western blot analysis of the steady-state levels of wild-type APOBEC3G and D128K mutant of APOBEC3G in the absence of any Vif or presence of HIV-1, SIVmac239 (SIVmac), and HIV-2 Vif proteins. The APOBEC3G proteins were detected by using an anti-myc tag antibody, and the tubulin proteins were detected with an anti-tubulin antibody to normalize the amounts of cell lysates analysed. Taken from reference 54 (No permission required to reprint).
23 1.1.2.9 VIRION ENCAPSIDATION OF APOBEC3G
Both the N- and C-terminals of APOBEC3G are required for encapsidation of
APOBEC3G into nascent virions. APOBEC3G mutants lacking the 67 N-terminal or 39 C-terminal amino acids fail to incorporate into virions 55 . Navarro and
colleagues 56 discovered that virion encapsidation was primarily mediated via the zinc coordinating and RNA binding residues of the N-terminal cytidine deaminase (CD1) domain. Mutation of the zinc coordinating Cys residues, Cys97 and Cys100, and the two highly conserved aromatic residues within the CD1 that mediate RNA binding
(Phe70 and Tyr91), but not the corresponding amino acids of the C-terminal cytidine deaminase (CD2) domain (Cys288, Cys291, Phe262 and Phe282), prevented encapsidation (Figure 1.14). The stable expression of the zinc coordinating mutants was reduced 2- to 4-fold compared to other mutants suggesting they also play a structural role 56 . The requirement of the APOBEC3G CD1 domain for APOBEC3G
encapsidation is conserved in HIV-1, human T cell leukemia virus type 1 (HTLV-1)
and murine leukemia virus (MuLV) 56 . The first linker region of APOBEC3G may also play a role in virion encapsidation as significant reductions in APOBEC3G encapsidation has been observed with mutants lacking amino acids 104-156 57 .
Virion encapsidation of APOBEC3G is highly dependent on the gag nucleocapsid
(NC) domain 57-62 . Indeed, APOBEC3G is able to interact specifically with gag
proteins produced by every retrovirus and endogenous retroelement examined so far
and also with the HBV core antigen (for review see Cullen 63 ). Specifically, the N- terminal 11 amino acids of the p7 region of NC are required for APOBEC3G encapsidation 57 . It is unclear if APOBEC3G binds directly to NC; as such
24 interactions only occur in the presence of RNA 59 . However, the RNA is unlikely to bridge the APOBEC3G-gag interaction as APOBEC3G-gag complexes can be immunoprecipitated from cell lysates following RNase treatment 61 . The requirement of gag for virion encapsidation of APOBEC3G is highlighted by MuLV, a simple retrovirus that is largely unaffected by expression of the catalytically active cognate murine APOBEC3, despite the fact it does not encode a vif protein (or any auxiliary protein), because its gag protein is unable to bind murine APOBEC3 and therefore does not encapsidate APOBEC3 64 .
Figure 1.14 Mutations of the zinc coordinating (Cys97 andCys100, 1CCAA) and RNA binding (Phe70 and Tyr91, 1FYAA) residues of CD1 but not of the CD2 (2CCAA & 2FFAA) domain reduces HIV-1 encapsidation of APOBEC3G . Virions were generated with ∆vif NL4-3 ( ∆vif ) and the mutated APOBEC3G-HA expression vector indicated above each lane. Taken from reference 56 (reprinted by permission from Elsevier Science) [ Virology , 2005;333:374-86].
Thus, it appears that APOBEC3G exploits the key role of gag to recruit the viral
RNA genome into virion particles to infiltrate virus particles. Therefore, by utilising a relatively non-specific mechanism of encapsidation, retroviruses are unlikely to develop specific escape mutations that prevent APOBEC3G encapsidation and may
25 explain why lentiviruses have evolved a gene whose sole function is to remove
APOBEC3G from the cell 59 .
1.1.2.10 ANTIVIRAL DOMAINS OF APOBEC3G
The duplicated active sites of APOBEC3G mediate distinct antiviral mechanisms.
Newman and colleagues 65 discovered using APOBEC3G mutants that single point mutations in the CD2 domain (H257R, E259Q, C288S and C291S) significantly decreased DNA-mutating activity compared to both wild type APOBEC3G and
APOBEC3G mutants harbouring corresponding CD2 domain mutations (H65R,
E67Q, C97S, and C100S) (Figure 1.15A). The antiviral activity of these CD1 and
CD2 domain mutants however was similar to that of wild type APOBEC3G and significant reductions in antiviral activity were only observed in the APOBEC3G mutants that harboured point mutations in both the CD1 and CD2 domains (Figure
1.15B). In contrast, Navarro and colleagues 56 demonstrated that mutation of the
catalytic residue of the CD2 domain (Glu259) but not that of the analogous residue
of the CD1 domain (Glu67), is sufficient to significantly diminish antiviral activity
of APOBEC3G. Although consistent with the results of Newman and colleagues 65 ,
the CD2 mutant had selectively lost cytidine deaminase activity. Thus, the CD2
domain of APOBEC3G is necessary for the DNA deaminase activity of APOBEC3G
and the CD1 domain exerts a DNA deaminase-independent antiviral function.
Consistent with the premise that the duplicated active sites of APOBEC3G function
independently, two separate domains of AID are independently responsible for
somatic hypermutation and class switch recombination. Furthermore, catalytically
26 inactive APOBEC3G mutants are as effective at inhibiting hepatitis B DNA synthesis as wild type APOBEC3G and despite incorporation of APOBEC3G into
HBV virions, C to U mutations were not detected 66 .
Figure 1.15 The Cytidine Deaminase Activity of APOBEC3G Is Not Required for an Antiviral Effect. (A) The ability of wild-type or mutated APOBEC3G proteins to mutate E. coli DNA. APOBEC3G-expressing vectors were transformed into bacteria, and the resulting induction of rifampicin resistance was quantitated by counting rifR colonies (relative to viable cell counts). The mutation rates for the mutant APOBEC3G proteins are plotted as a percentage of that seen with wild- type protein (set at 100%). Error bars represent the standard deviation. The lower panel shows immunoblots confirming comparable expression of the mutant APOBEC3G proteins in bacteria. (B) Antiviral function of APOBEC3G mutant proteins. Vif-deficient HIV-1 was produced in 293T cells transiently transfected with APOBEC3G expression vectors. Virus-containing supernatants were harvested at 24 hr and quantitated with a p24Gag ELISA. Normalized inocula were used to infect the C8166-CCR5/HIV-LTR CAT indicator cell line and the antiviral activity of each mutant protein is presented as a percentage of that seen for wild-type APOBEC3G. Error bars represent the standard deviation. The lower panel is an immunoblot confirming expression of the mutant APOBEC3G proteins in producer cell lysates. Taken from reference 65 (reprinted by permission from Elsevier Science) [ Curr Biol , 2005;15:166-70].
27 The trinucleotide target specificity exhibited by APOBEC3G (CC C) and
APOBEC3F (TT C) is governed by the CD2 domain. A chimeric APOBEC3F protein in which the CD2 domain was replaced by either the CD2 domain of APOBEC3G or the single cytidine deaminase domain of APOBEC3C exhibited the sequence specificity of the CD2 domain 67 . Interestingly however, chimeric APOBEC3F proteins in which the N- or C-terminal halves of the APOBEC3F CD2 domain were substituted by corresponding portions from APOBEC3C exhibited a dinucleotide specificity (C/TC C) quite distinct from that of either APOBEC3F or APOBEC3C
(TC/T C), yet similar to that of APOBEC3G.
28 1.1.3 VIRAL INFECTIVITY FACTOR (VIF )
1.1.3.1 HIV ACCESSORY /R EGULATORY GENES
Although the life cycle of lentiviruses such as HIV-1 is the same as for other retroviruses, lentiviral genomes are unique among retroviruses in that they encode six regulatory/accessory proteins involved in regulation of viral gene expression and infectivity (Figure 1.16) ( Tat, Rev, Vpu, Nef, Vpr, and Vif ). These proteins are essential for viral pathogenesis in vivo but are often not required for viral replication/infectivity in vitro (for reviews, see Emerman & Malim 68,69 and Anderson
& Hope 69 ).
Figure 1.16 Schematic representation of the HIV-1 genome. Blue – structural proteins; Yellow - accessory proteins; Pink - regulatory proteins; Grey – transcriptional start sites.
Briefly, transactivating transcription factor ( Tat ) regulates high level transcription of the proviral DNA mostly at the level of transcriptional elongation rather than initiation 70,71 . Regulator of viral protein synthesis ( Rev ) binds to a cis -acting RNA target ( Rev response element) present in all unspliced viral transcripts and targets them for nuclear export. Viral protein U ( Vpu ) and negative factor ( Nef ) cooperatively down regulate surface expression of the primary HIV-1 receptor,
CD4 +; Vpu binds to CD4 in the endoplasmic reticulum and promotes its proteolysis
29 by recruitment into the ubiquitin-proteasome pathway while Nef removes CD4 + from
the cell surface by accelerating endocytosis through clathrin-coated pits. Viral
protein R ( Vpr ) helps to maximise virus production by preventing infected cells from proliferating, arresting the cells in the G 2 phase of the cell cycle, the phase in which
the viral long terminal repeat (LTR) is more active72 . The role of the virion infectivity factor ( vif ) had been the least understood of the HIV-1 proteins, until
Sheehy and colleagues 11 isolated APOBEC3G, the cellular target of vif .
1.1.3.2 ROLE TO VIF
Vif is a highly basic, 23 kDa protein 73 that functions to prevent the encapsidation of
APOBEC3G into virions for degradation via the proteasomal pathway 46,74-80 . Early
researchers had noted that ∆vif HIV-1 virions had reduced infectivity (approximately
1000-fold) despite the production of similar amount of viral particles as wild-type
(wt) HIV-1 virions 81,82 . This was not due to impaired cellular entry or block in the early replication stages 83 but rather the impaired kinetics and extent of provirus DNA production 84-87 .
The requirement of vif for efficient HIV-1 replication was found to be cell type
dependent, required for HIV-1 replication in the CD4 + T-cell lines CEM and H9 as well as in peripheral blood T lymphocytes, but dispensable for replication in the
SupT1, C8166, and Jurkat T-cell lines (Figure 1.17). This suggested that vif can
compensate for cellular factors required for production of infectious virus particles
that are present in some cell lines such as SupT1, C8166, and Jurkat but are absent in
others such as CEM and H9 as well as peripheral blood T lymphocytes 82,88 . Indeed,
30 work by Simon and colleagues 9 and Madani & Kabat 10 utilising heterokaryons of permissive and non-permissive cells, demonstrated that HIV-1 virions produced from the heterokaryons are fully infectious, strongly supporting the conjecture that the natural targets of HIV-1 contain a potent endogenous inhibitor of HIV-1 replication that is overcome by vif .
Figure 1.17 Replication of vif-positive and vif-negative HXB2viruses in CD4 + human T-cell lines. Reverse transcriptase activity in the supernatants of T-cell cultures transfected with 10 µg of pHXB2 (open circles) or pHXB2 ∆vif (solid circles) plasmid DNA. Taken from reference 82 (reprinted by permission from American Society for Microbiology) [ J Virol , 1992;66:6489-95].
Through cDNA subtraction assays utilising cDNA from a non-permissive cell line
(CEM) and a permissive subclonal cell line (CEM-SS), Sheehy and colleagues 11 demonstrated that CEM15, a novel cellular protein, was abundantly expressed in non-permissive cells and absent or minimally expressed in permissive cells, and its ectopic expression in permissive cells renders them non-permissive. Analysis of the amino acid sequence of CEM15 revealed significant sequence homology to mammalian APOBEC1 and was subsequently termed APOBEC3G.
31 1.1.3.3 VIF -MEDIATED DEGRADATION OF APOBEC3G
Vif functions at several levels to prevent virion encapsidation of cellular
APOBEC3G, preventing APOBEC3G-mediated cytidine deamination of the progeny virions within the target cell. Vif binds APOBEC3G and sequesters APOBEC3G, excluding it from virion encapsidation, promotes degradation of the vif -APOBEC3G complex via the proteasomal pathway 49,74-76 and impairs the stability and translation of APOBEC3G mRNA 46 . Thus, vif functions to remove APOBEC3G from both nascent virions and virus producing cells.
The vif proteins of HIV-1, HIV-2 and SIV agm contain a highly conserved region with homology to the SOCS-box motif of cellular proteins that bind to Elongin B/C heterodimers. Indeed, coimmunoprecipitation and mass spectrometry revealed vif
binds Elongin B (EloB), Elongin C (EloC) and Cullin 5 (Cul5), core subunits of an
E3 ubiquitin ligase complex that promote the polyubiquitination of proteins, a post-
translational modification that controls the activity, localisation and proteasomal
degradation of many cellular proteins (Figure 1.18A). Several studies have
demonstrated that coexpression of vif and APOBEC3G in 293T cells result in the
polyubiquitination and decreased expression of both APOBEC3G and vif 74,78,79,89 .
The half-life of APOBEC3G in the presence of vif (1-2 minutes) indicates that
APOBEC3G is the most rapidly degraded protein studied to date 75 . In addition, Cul5 mutants and proteasomal inhibitors blocked the ability of vif to induce APOBEC3G degradation (Figure 1.18B). Therefore, vif is similar to F-box proteins, in that it is autoubiquitinated within its own E3 ligase complex. Thus a reciprocal relationship exists wherein vif and APOBEC3G influence steady state levels of each other.
32
A B
Figure 1.18 HIV-1 vif interacts with cellular proteins Cul5, ElonginB and ElonginC (A) to promote the polyubiquitination and subsequent proteasomal-mediated degradation of vif - APOBEC3G complexes (B). (A) Coimmunoprecipitation of cellular proteins with Vif -HA. Cell lysates from HXB2-or HXB2 Vif HA infected H9 cells were immunoprecipitated with HA antibody, followed by SDS–polyacrylamide gel electrophoresis (PAGE) and silver staining. The identification of Cul5, Elongin B, and Elongin C was achieved by mass spectroscopic analysis; these proteins were not detected in the control HXB2 samples; kDa – kilodaltons. Taken from reference 77 (reprinted by permission from American Society for the Advancement of Science) [Science, 2003;302:1056-60]. (B) 293T cells were transfected with pNLA1. Vif -FLAG (10 µg), pAPOBEC3G:HA (5 µg), and pRbG4-His6-myc-Ub (5 µg) vectors and treated with chloroquine (lysosomal inhibitor) or MG132/ β- lactone (proteasomal inhibitors) for 6 h before lysis. APOBEC3G and Vif were detected by Western blotting with anti-HA and anti-Vif antibodies, respectively. Taken from reference 79 (No permission required to reprint).
As mentioned, vif proteins contain a highly conserved SOCS-box motif required for binding to EloBC heterodimers and the subsequent proteasomal degradation of
APOBEC3G. This motif consists of approximately 25-40 amino acids and contains an N-terminal BC-box sequence and a C-terminal PPLP motif. The BC-box sequence of HIV-1 vif ( 145SLQYLAL 151) is the most conserved sequence among lentivirus vif proteins and differs from the BC-box of other viral and cellular proteins by containing an alanine at position 6 instead of cysteine. In addition to binding
33 EloBC heterodimers, dependent on leucine at position 7, the BC-box motif also binds Cul5. Precipitation of Cul5 with vif was completely abolished when wild type
vif was replaced by vif mutant ∆SLQYLAL. Specifically, it was found that it is the
alanine for cysteine substitution at position 6 of the BC-box motif that distinguishes
lentiviral vif from other viral and cellular BC-box motifs, in addition to the leucine at
position 2, which allows vif to bind to Cul5 79 . However, it was also found that the
precipitation of vif with Cul5 was significantly impaired for the vif mutant
C114/133S, in which cysteines 114 and 133 are substituted for serine. The cysteine molecules lack significant homology to other cullin-binding proteins and therefore are unlikely to be directly involved in binding; rather they may be important for protein conformation or other as yet unknown functions 79 . Such conclusions are
consistent with conflicting results from Kobayashi and colleagues 90 . They found that vif C114S and C133S mutants were able to bind Cul5 and were able to form
APOBEC3G-vif -EloBC-Cul5 complexes, however such complexes had lost their E3 ligase activity towards APOBEC3G (Figure 1.19).
The role of the C-terminal 161 PPLP 164 domain is unclear. Reports from Yang and
colleagues 91,92 indicate that the proline/leucine-rich domain is required for vif function by allowing vif multimerisation. Vif , like most HIV-1 proteins, possesses a strong tendency to self-association and is a requirement for their functionality (for review, see Frankel & Young 93 ). Yang and colleagues 92 demonstrated using synthesised overlapping 15-mers covering the entire vif sequence that vif -derived
peptides containing the 161 PPLP 164 domain inhibited the vif -vif interaction and that
deletion of the domain significantly impaired the capability of vif proteins to interact
with each other.
34
Figure 1.19 Vif C114S and C133S mutants are able to bind Cul5 (A) but fail to ubiquitinate APOBEC3G (B) despite similar levels of vif, EloBC and Cul5 expression (C). (A) Hi Five cells were co-infected with recombinant baculoviruses expressing His-vif (WT or mutants), myc-Cul5, EloB, EloC, and Rbx1. The lysates were immunoprecipitated with anti-vif mAb and analysed by immunoblotting with anti-c-myc mAb ( top panel ) and anti-vif serum ( middle panel ). Cell lysates were also analysed by immunoblotting with anti-c-myc mAb ( bottom panel ). Vif WT as well as C114S and C133S mutants could bind to Cul5 through EloB and EloC ( lanes 1–3, respectively), but the SLQ144/146AAA mutant did not ( lane 4 ). (B) An in vitro ubiquitin ( Ub ) conjugation assay was performed with a vif -BC-Cul5 complex (WT, C114S, or C133S). GST-ubiquitin-conjugated APOBEC3G was specifically detected as a ladder ( arrows ) only in a WT vif -BC-Cul5 complex ( lane 3) but not in C114S and C133S vif -BC-Cul5 complex ( lanes 4 and 5, respectively). (C) Hi Five cells were co-infected with recombinant baculoviruses expressing His-Vif (WT, C114S, or C133S), Cul5, EloB, EloC, and Rbx1, and His-Vif protein was purified with NTA beads. Purified His-Vif and associated proteins were visualized by immunoblotting with the appropriate antibodies. Taken from reference 90 (No permission required to reprint).
1.1.3.4 VI F ENCAPSIDATION
Vif is a cytosolic protein that, in the absence of APOBEC3G, associates with the plasma membrane via an interaction with gag 94,95 that allows co-encapsidation of both vif and gag into virions 96,97 . Vif specifically associates with the Pr55 gag precursor, with no interaction with the mature capsid protein. Deletion of the gag residues H421-T470, which corresponded to the C-terminal region of nucleocapsid
(NC), including the second zinc finger, the intermediate spacer peptide sp2 and the
N-terminal half of the p6 domain, significantly decreased vif encapsidation efficiency, and complete deletion of NC abolishes vif encapsidation. In vif , four discrete gag -binding sites were identified, within residues T68-L81 (site I) and W89-
P100 (site II) in the central domain, and within residues P162-R173 (III) and P177-
35 M189 (IV) at the C terminus. Substitutions in site I and deletion of site IV were detrimental to vif encapsidation, whereas substitution of basic residues for alanine in sites III and IV had a positive effect 96 .
1.1.3.5 REGULATION OF VI F
Phosphorylation of vif plays an important role in regulating HIV-1 replication and infectivity. Indeed, several HIV-1 proteins, including p24 Gag , p17 Gag , Vpu , Rev
and Nef have been shown to be regulated, at least in part, by phosphorylation 98-102 . In
vitro phosphorylation studies utilising 32 P-labelled vif and protease digestion followed by HPLC resolution and radioactive sequencing revealed vif phosphorylation is restricted to the C-terminus amino acids, Ser 144 , Thr 155 and
Thr 188 103,104 . Despite Thr 155 and Thr 188 being contained within the R/KXXS*/T* and R/KXXXS*/T* recognition motifs used by serine/threonine protein kinases, such as cGMP-dependent kinase and protein kinase C, inhibitors of such kinases failed to inhibit vif phosphorylation, questioning the role of these phosphorylation
sites. Indeed, Thr 188 is highly conserved amongst HIV-1 isolates but not among other lentiviruses while Thr 155 is not highly conserved. In contrast, substitution of
Ser 144 for Ala 144 , which is contained within the highly conserved BC-box motif
145SLQYLAL 151 and is critical for vif function, resulted in loss of vif function and
over 90% inhibition of HIV-1 replication (Figure 1.20) 103 . The kinase responsible for
phosphorylation of Ser 144 was not identified.
Subsequent studies from the same group 104,105 identified two additional
phosphorylation sites, Thr 96 and Ser 165 , that were phosphorylated by p44/42
36 mitogen-activated protein kinase (MAPK, also known as extracellular protein kinase
1 (ERK1) and ERK2) despite them not being located in the PX(S/T)P or (S/T)P consensus motifs for MAPK substrates. MAPK play critical roles in regulation of cell proliferation and differentiation in response to mitogens and a wide variety of growth factors and cytokines (reviewed by Marshall 106,107 and Seger & Krebs 107 ).
Thus, phosphorylation of vif by MAPK may contribute to the activation of HIV-1 replication by mitogens and other extracellular stimuli.
Figure 1.20 Phosphorylation of HIV-1 proteins by MAPK. In vitro kinase assays were performed using recombinant p42 MAPK. MAPK immunocomplexes were isolated from cell lysates by using anti-ERK1 and anti-ERK2. MAPK was incubated with 2 mg of each indicated recombinant HIV-1 protein for in vitro kinase assays performed in the presence of [g-32P]ATP. The results of phosphorylated amino acid analysis are shown on the right. The positions of phosphorylated Ser, Thr, and Tyr (pSer, pThr, and pTyr) markers are indicated. Phosphorylated amino acids detected in the recombinant HIV-1 proteins comigrated with pSer and pThr but not with pTyr. Taken from reference 104 (No permission required to reprint).
1.1.3.6 VIF -APOBEC3 BINDING
As mentioned in section 1.1.3.3, in addition to facilitating the formation of vif -
APOBEC3G-Cul5-EloBC E3 ligase complexes that promote the degradation of
APOBEC3G through the ubiquitination and proteasomal pathway, vif also prevents encapsidation of APOBEC3G by simply forming vif -APOBEC3G complexes that are sufficient to inhibit deaminase activity.
37 Ectopic coexpression of APOBEC3G and vif in E.coli allowed Santa-Marta and
colleagues 50 to investigate the role of proteasomal degradation in inhibiting
APOBEC3G activity as E.coli lack ubiquitin-proteasomal machinery. Using this approach, it was found that expression of vif was sufficient to inhibit APOBEC3G deaminase activity. In addition, substitution of the vif C114 residue, which is required for E3 ligase activity 90 , failed to restore APOBEC3G deaminase activity,
clearly demonstrating that vif -APOBEC3G binding is sufficient for inhibition of
APOBEC3G deaminase activity. This is consistent with confocal observations
presented in Figure 1.21, demonstrating that APOBEC3G can be stably expressed in
the presence of excess vif and that viruses produced from cells stably coexpressing
APOBEC3G and vif are fully infectious 108 .
Figure 1.21 Stable coexpression of APOBEC3G and vif . Cells were stained with a rabbit polyclonal antibody to APOBEC3G (A & D) and a monoclonal Vif antibody (B & E). APOBEC3G was visualized using a Texas red-conjugated secondary antibody; Vif was visualized with a Cy2- conjugated secondary antibody. Panels C and F are merged images of panels A & B and D & E, respectively. Arrow heads are defined as follows: white = APOBEC3G: Vif -double-positive cells; yellow = Vif -negative cells; red = cells exhibiting nuclear and cytoplasmic staining for Vif . Taken from reference 108 (No permission required to reprint).
38 While it is clear that the charge-changing D128K APOBEC3G polymorphism
50-53 selectively facilitates APOBEC3G to bind HIV-1 vif or SIV agm vif , respectively , the corresponding binding site(s) on vif are unclear. Using competitive phage-ELISA assays to detect specific interactions between phage-displayed APOBEC3G and overlapping clade B vif 15-mers spanning the entire vif protein, significant inhibition of APOBEC3G-vif complex formation was observed with peptides spanning amino acids 13-39, 73-99, 133-147 and 169-192 at salt concentrations that favour low energy interactions 50 . Using high salt concentrations that select for strong
APOBEC3G-vif peptide interactions, only vif peptides spanning amino acids 85-99 and 169 to 192 significantly inhibited APOBEC3G-vif complex formations, implicating both the C- and N-terminal regions (Figure 1.22) 50 . Consistent with these results, work by Simon and colleagues (2005) using a panel of vif mutants containing naturally occurring mutations, found expression of vif mutants harbouring K22E,
S32P, Y40H, E45G, F115S, G138R or L150P mutants failed to maintain HIV-1 infectivity in 293T cells coexpressing APOBEC3G. Furthermore, specific amino acids within the C-terminus were found to selectively neutralise APOBEC3G and
APOBEC3F. The K22E vif mutant retained partial activity against APOBEC3F, but was inactive against APOBEC3G and conversely, Y40H and E45G vif mutants neutralised APOBEC3F as efficiently as wild type vif but failed to neutralise
APOBEC3G.
39
Figure 1.22 Vif peptides comprised of vif amino acids 85-99 and 169 to 192 significantly inhibit interaction between phage-displayed APOBEC3G and vif . Competitive ELISA of phage-displayed APOBEC3G against vif protein using HIV-1 consensus B vif (15-mer) peptides covering the complete vif sequence. Recombinant vif protein was added to a 96-well plate. Phage-displayed APOBEC3G was preincubated with 100 µM of independent peptides in low or high ionic buffer. After incubation, excess phages were washed off, and HRP-conjugated anti-M13 monoclonal antibody was used for detection. The results were measured by optical density at 405 nm. Taken from reference 50 (No permission required to reprint).
40 1.1.4 CLINICAL SIGNIFICANCE
1.1.4.1 APOBEC3G GENETIC VARIATION
Two studies to-date have examined the clinical significance of APOBEC3G genetic variation; each examining the influence of APOBEC3G genetic variation on progression to AIDS. The first study, conducted by An and colleagues 109 identified seven single nucleotide polymorphisms (SNPs) utilising nonisotopic RNA cleavage assays and DNA sequencing targeting the putative 5’ regulatory region, exonic regions, intron/exon junctions, intron 1 and the 3’ untranslated region of the
APOBEC3G gene from 92 European Americans and 92 African American individuals. The genotype of each SNP was then determined for 2,430 individuals enrolled in the United States-based HIV/AIDS prospective cohorts, 643 participants in the Swiss HIV Cohort Study (SHCS) and 129 healthy Chinese individuals and haplotypes deduced by the expectation maximisation algorithm. Comparing allele, genotype and haplotype frequencies in seropositive individuals to seronegative individuals revealed no association between APOBEC3G genetic variation and HIV-
1 transmission. Regarding AIDS progression, the 186R APOBEC3G variant, common amongst African Americans but rare amongst European Americans (37% allele frequency versus 3%, respectively) was significantly associated with both rapid progression to AIDS and death and more rapid decline of CD4 + T-cell slope than individuals homozygous for the H185 APOBEC3G variant (Figure 1.23A).
However, in vitro studies did not reveal a significant difference in antiviral activity between the H186R APOBEC3G variants (Figure 1.23B), although the influence of the 186 polymorphism on G→A mutation, which may promote the emergence of escape mutants, was not determined.
41 A B
Figure 1.23 The APOBEC3G 186R variant associated with accelerated progression to AIDS exhibits similar antiviral activity to the 186H variant. (A) Kaplan-Meier survival curves of HIV-1 progression to AIDS-1987 after seroconversion for the three genotypes of APOBEC3G-H186R in African Americans. RH and P values are from an adjusted Cox model analysis, and log rank P value compares the Kaplan-Meier curves. (B) 293T cells were transfected with R9 HIV-1 or with the vif - defective R9 ∆vif proviral clone in the presence of empty vector, wt hAPOBEC3G (H186R), or 186R APOBEC3G-expressing vector. Supernatant was filtered, and viral titer was scored by infecting HeLa-CD4-LacZ reporter cells not expressing APOBEC3G. Virion infectivity is normalized for particle amount, as measured by RT activity assay. Taken from reference 109 (reprinted by permission from American Society for Microbiology) [ J. Virol . 2004;78:11070-76].
The second study, conducted by Do and colleagues 110 identified 42 polymorphisms,
of which 29 had allele frequencies greater than 1% and 14 were novel from 327
clade B infected French individuals from the Genetics of Resistance to
Immunodeficiency Virus (GVIR) cohort and 446 healthy controls. Similar to the
previous study by An et al 109 , this study failed to identify any significant associations
between APOBEC3G polymorphisms or deduced haplotypes or haploblocks on
progression to AIDS. These studies reveal APOBEC3G to be a highly conserved
protein amongst Caucasian individuals, with only two non-synonymous
polymorphisms identified at very low frequencies. Thus, among Caucasian
individuals, it is likely that viral genetic variation contributes greater to
hypermutation and its clinical consequences than host genetic variation.
42 1.1.4.2 VI F GENETIC VARIATION
Vif genetic variation has been associated with long-term non-progression status although whether these associations manifest through hypermutation is unclear.
Yamada and Iwamoto 111 observed nonsense mutations in the vif open reading frame in 11/30 (37%) and 1/229 (0.4%) clones from seven long-term non-progressors and seven progressors, respectively, while Wang et al 112 noted tryptophan to stop codon mutations were a consistent feature in longitudinal samples isolated from a long-term non-progressor over eight years. Tryptophan to stop codon mutations are characteristic mutations mediated by APOBEC3G and therefore, the associations between nonsense mutations and long-term non-progression described above are likely to represent associations between hypermutation and long-term non- progression. Indeed, comparison of three full-length clade B HIV proviral clones from a long-term non-progressor to the hxb2 clade B reference sequence revealed extensive G→A hypermutation and numerous in-frame stop codons all due to tryptophan to stop codon mutations 113 . However, whether the defective vif sequences and hypermutation associated with long-term non-progression is the cause or an effect is unclear.
Hassaine and colleagues 114 identified significant correlations between vif amino acid
132 and progression status. S132 was present significantly more frequently in long- term non-progressors with low viral loads than long-term non-progressors with high viral loads. In vitro studies revealed that the replicative capacity of the S132 variant was reduced 5.1 ± 0.5 fold compared to the R132 variant only in non-permissive cells, with similar replicative capacity observed in permissive cells. Thus, the S132
43 vif variant associated with long-term non-progression status is likely to mediate this
effect through hypermutation.
44 1.1.5 HYPOTHESIS & AIMS
1.1.5.1 HYPOTHESIS
We hypothesised that G→A hypermutation conforming to the APOBEC3G and/or
APOBEC3F sequence context would be evident in a minority of HIV-1 proviral
DNA and would be associated with reduced pre-treatment viremia. In addition,
G→A hypermutation would be associated with host (APOBEC3G, and/or
APOBEC3F depending on which appears to be the dominant contributor to G→A hypermutation) and viral ( vif ) genetic variation.
1.1.5.2 AIMS
1. To estimate G→A hypermutation in HIV-1 proviral DNA at the population
level to address whether APOBEC3G or APOBEC3F is the dominant
contributor to HIV-1 hypermutation.
2. To sequence the entire gene of the causative APOBEC3 enzyme to determine
its influence on G→A hypermutation.
3. To determine whether vif genetic variation is associated with G→A
hypermutation.
4. To determine whether G→A hypermutation influences pre-treatment viremia.
45 1.2 MATERIALS & METHODS
1.2.1 PATIENT SELECTION
One hundred and thirty six adult antiretroviral-naïve HIV-infected patients were selected from the Western Australian HIV cohort 115 representing a predominantly
Caucasian (84%) male (88%) study population who had acquired HIV infection through sexual contact (83%). To be included in the present study, >1000 nucleotides of proviral HIV sequence assigned as clade B (see below) were required per sequence. All clade B sequences were utilised in the construction of the population consensus HIV-1 sequence in this study. For the investigation of G →A substitutions, nine sequences having fewer than 100 nucleotide substitutions from consensus were excluded from the analysis, leaving n=127 cases.
1.2.2 HIV-1 CLADE ASSIGNMENT
HIV sequences were analysed by phylogenetic analysis using Molecular
Evolutionary Genetic Analysis, version 3 (MEGA 3.0, http://www.megasoftware.net ) as follows. The nucleic acid sequences for the
individual HIV-1 protein products (e.g., p17, p24, reverse transcriptase) were
extracted from the database. These were combined with 6 to 15 sequences from each
of clades A, B, C, D, and F as well as the sequence for HXB2 and the M-group
ancestral sequence; these reference sequences were downloaded from the Los
Alamos HIV sequence database. Phylogenetic trees were constructed using the
parameters 1) neighbour-joining tree inference, 2) pairwise deletion, and 3) Kumara
2-parameter substitution model. The resulting trees were then rooted on the M-group
46 ancestral sequence and inspected. Most of the sequences clearly sorted with reference sequences of a particular clade and these were assigned as belonging to that clade. Sequences that did not clearly belong to a specific clade were assigned as unknown. A final assignment of clade was made based on the assignments for all of the proteins. If all of the protein products for a particular patient were unambiguously assigned as the same clade, that sample was assigned as that clade. If all protein products were the same clade except for one protein product assigned as ambiguous, the consensus clade was again assigned to that sample. Samples where the proteins belonged to several clades were noted as such (e.g. AB, BC) and excluded from subsequent analysis.
1.2.3 AMPLIFICATION AND SEQUENCING OF HIV-1 PROVIRAL DNA,
MEASUREMENT OF PRE -TREATMENT HIV RNA LEVELS , HLA
AND CCR5 GENOTYPING
These were performed as previously described in Moore et al 116 . Patient DNA was extracted from blood samples using the QIAGEN DNA extraction kit according to the manufacturer’s instructions. The PCR conditions and primers used for the full- length amplification of the proviral HIV genome have been previously described in
Oelrichs et al 117 . Briefly, the first-round PCR was performed with the Expand Long
Templates PCR Kit (Boehringer Mannheim) to produce a 9kb amplified product.
This first round product was then used as a template for 13 individual nested PCR reactions using Taq polymerase (Boehringer Mannheim) to amplify the entire HIV genome from gag p17 to the 3’ LTR. First and second round PCR reactions were performed on ABI 9700 and 9600 thermocyclers. Successfully amplified PCR products were sequenced in the reverse and forward directions using
47 BigDyeTerminator ready reaction prism kits (v3). The samples were electrophoresed on the ABI 3100 Genetic Analyser, and sequencing data analysed using ABI software package, Seqscape Version 1.1. Mixtures (sites where more than one nucleotide was observed) were named according to the IUPAC standard. Sites that were unable to be assigned were designated as ‘N’ and excluded from the analyses.
Nucleotide sequence length within the study population averaged 6820 ± 1187 nucleotides (range 1329 – 8768 nucleotides).
1.2.4 ANALYSIS OF G→→→A SUBSTITUTIONS
To estimate G→A substitutions, individual HIV-1 proviral DNA sequences were aligned against the population consensus clade B sequence (n=136). Only G→A substitutions where the nucleotide at position +1 in the sample matched the corresponding nucleotide in the consensus sequence were examined (where GA, GC,
GG, and GT dinucleotides in the consensus sequence were observed as AA, AC, AG,
and AT in the sample sequence, respectively. Mixture nucleotide results obtained from chromatograph analysis (indicating the presence of mixed nucleotide populations) were assigned as missing values. To estimate ‘non-specific’ G →A hypermutation for each sequence (ie. without reference to the expected dinucleotide sequence context for APOBEC3F or APOBEC3G), we incorporated two previously used measures of hypermutation 11 , G →A preference (#G →A substitutions/#all mutations) and G →A burden (#G →A substitutions/# consensus G) into a single formula:
48 Non-specific G→A hypermutation score = (#G→A substitutions/# consensus G) (#mutations/#nucleotides sequenced)
This represents the proportion of all mutations that are G →A substitutions adjusted for the proportion of nucleotides sequenced that are G in the consensus sequence.
1.2.5 ANALYSIS OF APOBEC3G AND APOBEC3F TARGET MOTIFS
To investigate the contribution of APOBEC3G and APOBEC3F to hypermutation, we examined the dinucleotide sequence contexts of G→A substitutions using the following formulae:
3G-specific G→A substitution score = (#GG →AG substitutions/#consensus GG) (#G→A/#G)
3F-specific G→A substitution score = (#GA →AA substitutions/#consensus GA) (#G→A/#G)
These represent the proportion of all G →A substitutions that occurred in the GG
(APOBEC3G) and GA (APOBEC3F) contexts adjusted for the number of available
GG (APOBEC3G) and GA (APOBEC3F) dinucleotides.
49 1.2.6 ANALYSIS OF APOBEC3G- AND APOBEC3F-MEDIATED
HYPERMUTATION
We combined the hypermutation formula with either the 3G or 3F formula to identify sequences that had both an inordinate number of G→A substitutions relative
to other substitutions and a high preference for G→A substitutions specifically in the
sequence context targeted by either APOBEC3G or APOBEC3F as follows:
Consolidated 3G score = (#GG →AG/#consensus GG) (#mutations/#nucleotides sequenced)
Consolidated 3F score = (#GA →AA/#consensus GG) (#mutations/#nucleotides sequenced)
Thus, these represent the proportion of all G→A substitutions that occurred in the
GG (consolidated 3G score) and GA (consolidated 3F score) dinucleotide contexts, adjusted for the overall mutational rate.
Histogram and Q-Q plots of the consolidated 3G values (log 10 transformed) suggested a bimodal distribution with a normally distributed main group and a smaller group with higher values. The bimodal distribution was fitted by a mixture of two normal distributions using maximum likelihood, giving estimated distributions with means, SD of -0.277, 0.151 and 0.417, 0.151, respectively, and an estimated 9.4% of observations in the latter group. The presence of a mixture was highly significant (p<0.00005, likelihood ratio test). The likelihood of belonging to the upper group was higher for the largest 12 observations, with one likelihood ratio
50 of 8 and the rest greater than 140. Hierarchical cluster analysis using between- groups- and centroid-linkage both give two clear groups with n=115 and n=12 as does k-means clustering. The non-hypermutated (NH) group is approximately normal. The corresponding analysis of the non-APOBEC3G-mediated hypermutated cases suggests three cases with inordinate consolidated 3F values.
1.2.7 APOBEC3G ALLELE FREQUENCIES
From 8 participants with the highest G →A hypermutation scores and a pooled DNA control sample (n=187 Caucasian individuals) 118 , the entire APOBEC3G gene, including 2kb 5’ of the transcription start site and 1.0 kb 3’ of the transcription stop site, was amplified as 2 products of approximately 6.5kb (3’ half) and 8kb (5’ half), size. For amplification of each product, 1 µL of DNA was amplified in a volume of
50 µL containing 10X Hi-Fi PCR buffer, 2mM MgSO4 (1.5mM MgSO4 for 3’ half),
0.8mM dNTP mix, 100nM of each forward and reverse primer, 1U of PlatinumTaq
Hi-Fi ® and 2% DMSO (1.5% for 5’ half) under the following conditions: 94 °C for
30 s, followed by 35 cycles of 94 °C for 30 s, 63 °C for 30 s (60 °C for 5’ half) and
68 °C for 9 m. The PCR products were then purified using Exosap ® according to the manufacturer’s protocol and sequenced using overlapping primers (Supplementary
Figure 1 and Supplementary Table 1) and the Big Dye Terminator kit, version 3.3
(PE, Applied Biosystems, USA). The allele frequencies of the pooled DNA sample were determined by chromatograph relative peak height and compared to the allele frequencies of the 8 patients harbouring the HIV-1 proviral sequence with the greatest G →A hypermutation scores. Allele frequencies estimated from sequencing of pooled DNA have previously been shown to correlate well with allele frequencies
51 measured by other methods and provide an accurate, cost-effective method for determining allele frequencies 118,119 . To determine the lower limit at which alleles
could be detected by sequencing pooled DNA, PCR products homozygous for
alternative alleles of a thymidine kinase 2 SNP were mixed at various proportions
and sequenced. The results, presented in Supplementary Figure 1.31s, suggest that
the lower limit of allele detection by sequencing is approximately 10-15%.
1.2.8 STATISTICAL ANALYSIS
Statistical analyses were performed using SPSS software (version 12.0.1, SPSS Inc.
USA). All values are expressed as mean ± standard deviation unless otherwise stated. Where data were normally distributed, ANOVA was used for group comparisons, with correction for multiple comparisons utilising the Bonferroni method where appropriate. For non-normal data, Mann-Whitney tests were performed. The presence of in-frame stop codons in vif amino acid sequences was determined by putative translation of the proviral DNA sequences. With reference to analyses of the determinants of plasma HIV RNA levels prior to antiretroviral therapy, HLA-B27 (n=7) and HLA-B58 (n=4) were also considered as covariates along with HLA-B57 (n=12) and the CCR5 ∆32 genotype (n=25).
52 1.3 RESULTS
1.3.1 ANALYSIS OF G→→→A SUBSTITUTIONS
We first sought to ascertain the population distribution of HIV-1 G →A substitutions, in order to identify hypermutated HIV-1 sequences within the context of natural in vivo sequence variation. The study was restricted to sequences identified as clade B by phylogenetic analysis, involving a study population of predominantly male (87%)
Caucasian (84%) patients with chronic HIV-1 infection. All participants were antiretroviral-naïve at the time of clinical assessment and collection of proviral HIV-
1 DNA for sequencing, as reflected in the distribution of HIV RNA/mL viral load
+ (mean ± SD, 4.903 ± 0.760 log 10 copies), CD4 T cell counts (380.6 ± 298.3 cells/mL) and percentage CD4 + T cells (19.3 ± 10.9%). To investigate G →A substitutions at the population level, we aligned each individual’s HIV-1 proviral sequence to the population consensus sequence, and for each sequence considered two measures of hypermutation as previously described 120 ; (1) the preference for
G→A substitutions relative to all other substitutions (defined by the proportion of all mutations that were G →A substitutions); and (2) the burden of G →A substitutions relative to the number of available consensus guanine nucleotides (defined by the proportion of consensus guanines that exhibited G →A substitutions). As expected, we found that G →A substitutions were the most common substitution observed
(median ± IQR; 21.0 ± 4.0% of all substitutions), and there was a highly significant correlation between G →A preference and G →A burden in the study population
(Spearman’s r=0.631, P<0.001), as shown in Figure 1.24.
53
A
A burden A → → → → G
G→→→A preference
Figure 1.24 G →→→A burden and G →→→A preference are highly correlated. Blue open circles – NH sequences; red shaded circles – HM-3G sequences; black open triangles – HM-3F sequences; *, † - see Materials and Methods sections “Analysis of APOBEC3G and APOBEC3F dinucleotide target motifs” and “Analysis of G →A substitutions” , respectively.
We then analysed appropriately transformed data to ascertain if the distribution of
G→A substitutions conformed to a model in which a proportion of sequences could be described as ‘hypermutated’ relative to natural sequence variation (see Methods).
Using various cluster analyses as well as analyses of population mixtures, these data were best described by a bimodal distribution rather than a normal distribution
(P<0.001), consistent with the hypothesis that a proportion of proviral DNA sequences exhibited HIV-1 hypermutation.
1.3.2 ANALYSIS OF APOBEC3G AND APOBEC3F DINUCLEOTIDE
TARGET MOTIFS
As mentioned previously, APOBEC3G and APOBEC3F are known to target specific single-stranded polynucleotide DNA motifs. Therefore, in order to further characterize and investigate APOBEC3-associated HIV-1 hypermutation, we further
54 examined the preference for G →A substitutions in the APOBEC3G ( GG) and
APOBEC3F ( GA) dinucleotide contexts. This was first accomplished by investigating the dinucleotide sequence context for G →A substitutions (see
Methods). Again, the population distribution of G →A substitutions indicated the presence of APOBEC3G-mediated G →A substitutions in a proportion of sequences, in that there was distinct bimodality in the distribution of GG→AG substitutions in the study population (Figure 1.25A, P<0.001). A small number of sequences were identified that exhibited an extreme preference for APOBEC3F-mediated G →A substitutions (ie. GA→AA), although this small group (n=3) could not be identified as a distinct data cluster (Figure 1.25B).
BA CCB
X G A : A A substitution A score* A substitution A score* G → → → → → → → →
3F-specific G 3F-specific G 3G-specific
† † non-specific HMG→→→A HM score non-specific G→→→A HM score
Figure 1.25 G→→→A substitutions in HIV-1 proviral DNA with inordinate G →→→A preference and G→→→A burden occur predominantly in the dinucleotide sequence contexts targeted by APOBEC3G ( GG) and, to a lesser extent, APOBEC3F ( GA). (A) G →A preference (# G →A substitutions/all mutations) was highly significantly correlated to G →A burden (#G → substitutions/#consensus G) (r=0.853, P<0.001). (B and C) The general G →A hypermutation scores were significantly correlated with the 3G-specific G →A substitution scores (r=0.318, P<0.001) but not with the 3F-specific G →A substitution scores (r=0.065, P=0.465). Red shaded circles – HM-3G sequences determined by mixed population analysis of the consolidated 3G score; black open triangles – HM-3F sequences determined by mixed population analysis of the consolidated 3F score; Blue open circles – NH sequences determined by mixed population analyses of the consolidated 3G and consolidated 3F scores; † - general G →A hypermutation score (#G →A substitutions/#consensus G)/(#mutations/#nucleotides sequenced); * - 3G-specific G →A substitution score (#GG →AG substitutions/#consensus GG)/(#G →A/#G); ‡ - 3F-specific G →A substitution score (#GA →AA substitutions/#consensus GA)/(#G →A/#G).
55 1.3.3 CLASSIFICATION OF APOBEC3G- & APOBEC3F-
HYPERMUTATED HIV-1 SEQUENCES
In order to formally identify sequences with evidence of APOBEC3G-mediated hypermutation (HM-3G), we then analysed the population distribution of HIV-1
G→A substitutions incorporating both (1) G →A preference relative to other
mutations, and (2) preference for G →A substitutions within the GG context (Figure
1.26, see Materials & Methods “Classification of APOBEC3G- and APOBEC3F- hypermutated HIV-1 sequences” ). Mixture models and cluster analyses consistently identified 12 sequences (9.4%) that fulfilled these criteria (P<0.001). As shown in
Table 1.1, these sequences exhibited a 1.9-fold preference for G →A substitutions on average, and a 3.1-fold increase in G →A burden compared with the remaining ‘non- hypermutated’ (NH) sequences (all P<0.001). In addition, 42.7 ± 8.7% of all G →A substitutions were in the GG context, compared with 17.7 ± 5.0% in the NH
sequences (P<0.001). The average proportion of consensus guanine bases that
demonstrated G →A substitutions in these hypermutated sequences (14.2%) was consistent with previous descriptions of hypermutated HIV-1 proviral DNA obtained from env gene sequences in vivo (16%) 20 and utilising ∆vif virions in vitro 65 .
Three additional sequences showed a strong preference for APOBEC3F-mediated hypermutation (HM-3F) using these criteria (Table 1), with 59.9 ± 11.3% of all
G→A substitutions occurring in the GA context, compared with only 26.1 ± 7.0%
and 32.1 ± 6.0% in the HM-3G and NH sequences, respectively (both P<0.001).
56 While G →A substitutions were the most common substitution observed in the 112
NH sequences, there was no significant association between the non-specific G →A hypermutation score and the 3G-specific G →A substitution score (Pearson’s r=0.093, P=0.331) or 3F-specific G →A substitution score (r=0.140, P=0.142).
Hence, it is unlikely that APOBEC3G or APOBEC3F-mediated DNA editing contribute significantly to the rate of G →A substitutions (identified by bulk PCR sequencing) within these non-hypermutated proviral DNA sequences.
20
15
10 frequency
5
0 -2 -1 0 1 2
consolidated 3G score (log 10 )
Figure 1.26 The consolidated 3G scores reflect a bimodal distribution . The bimodal distribution of the consolidated 3G scores (see Materials and Methods section “Analysis of APOBEC3G- and APOBEC3F-medaited hypermutation”) was fitted by a mixture of two normal distributions utilising maximum likelihood, giving estimated distributions with means, SD of -0.636, 0.348 (blue shaded bars; subsequently defined as non-hypermutated sequences, NH) and 0.964, 0.344 (red partially- shaded bars; subsequently defined as APOBEC3G-mediated hypermutated sequences, HM-3G), respectively. The presence of a population mixture was highly significant (P<0.0005, likelihood ratio test).
57 Table 1.1 Patient and sequence characteristics of patients harbouring putative APOBEC3G-, APOBEC3F-hypermutated and non-hypermutated HIV-1 proviral DNA Patient Characteristics NH HM-3G HM-3F n 112 12 3 CD4 + count/mL 367.2 (306.1) 491.3 (236.7) 421.3 (152.1) CD4 + (%) 18.5 (10.7) 24.7 (11.4) 28.3 (8.3) Viral load (log 10 copies/mL) 4.981 (0.747) 4.476 (0.50)* 3.721 (0.680)*
HIV-1 Sequence Characteristics
n nucleotides sequenced per patient 6906 (1016) 6571 (1549) 4591 (3182) number of non-G→A mutations 290 (66) 295 (97) 246 (129) number of G →A substitutions 75 (18) 210 (107) 110 (40) G→A rate (per 100 nucleotides) 1.08 (0.21) 3.4 (1.8) 3.3 (2.4) G→A substitutions: all substitutions 0.206 (0.029) 0.40 (0.123) 0.321 (0.049) G→A burden (% of consensus G’s) 4.6 (0.9) 14.2 (7.5) 14.9 (11.4) GG (% of G →A substitutions) 17.7 (5.0) 42.7 (8.7) 10.8 (5.2) GA (% of G →A substitutions) 32.1 (6.0) 26.1 (7.0) 59.9 (11.3) Hypermutation Scores † Non-specific G →A hypermutation -0.06 (0.06) 0.22 (0.11) 0.14 (0.08) 3G-specific G →A substitutions -0.21 (0.13) 0.19 (0.11) -0.45 (0.25) 3F-specific G →A substitutions -0.05 (0.08) -0.15 (0.11) 0.22 (0.06) Consolidated 3G -0.28 (0.15) 0.41 (0.16) -0.30 (0.16) Consolidated 3F -0.12 (0.11) 0.07 (0.15) 0.36 (0.15)
Numbers are expressed as mean (standard deviation) where appropriate; n – number; NH – non-hypermutated; HM-3G – APOBEC3G-mediated hypermutated sequences; HM-3F – APOBEC3F-mediated hypermutated sequences; GG, GA – percent of G →A substitutions that occurred in the GG, or GA dinucleotide contexts, respectively; * >95% significance level compared to NH sequences; † - see materials and methods, log10 transformed values; significance not determined for HIV-1 sequence characteristics or hypermutation scores as these were the basis of the classifications.
58 1.3.4 HIV-1 GENOMIC DISTRIBUTION OF G→→→A HYPERMUTATION
From the data presented in Figure 1.27, measures of general G →A hypermutation
(Figure 1.27A), APOBEC3G-specific G →A substitutions (Figure 1.27B) and
APOBEC3G-mediated hypermutation (Figure 1.27C) were distributed relatively evenly along the genome. Consistent with results from Yu and colleagues 121 , general
G→A hypermutation and APOBEC3G-mediated hypermutation scores in HM-3G sequences were significantly higher in pol than gag (P<0.001 and P=0.005, respectively), however in contrast, significantly lower in env than in pol (both
P<0.001). Furthermore, consistent with results from Wurtzer and colleagues 122 , the distance from the central polypurine tract was significantly negatively correlated with the general G →A hypermutation (r=-0.283, P=0.007), APOBEC3G-specific substitution (r=-0.250, P=0.020) and APOBEC3G-mediated hypermutation (r=-
0.297, P=0.006) scores.
1.3.5 ASSOCIATION OF VIF AMINO ACID POLYMORPHISMS AND
G→→→A HYPERMUTATION
We subsequently sought to investigate associations between vif amino acid polymorphisms and hypermutation. For such analyses, we performed a putative translation of available vif sequences (n=124) and examined vif polymorphism within the NH sequences ( vif amino acids 1-193) compared with the corresponding amino acid in the HM-3G sequences. HM-3F sequences were omitted from these analyses so that specific associations relevant to the APOBEC3G-vif interaction could be examined, as it has recently been reported that specific vif amino acid
59 polymorphisms differentially influence APOBEC3G versus APOBEC3F interaction 123 .
A
B
C
nucleotide position
Figure 1.27 General G →→→A hypermutation ((#G →→→A substitutions/#consensus G)/(#mutations/#nucleotides sequenced)), APOBEC3G-specific G →→→A substitutions ((#GG →→→AG substitutions/#consensus GG)/(#G →→→A/#G)) and APOBEC3G-mediated hypermutation ((#GG→→→GA substitutions/#consensus GG)/(#G →→→A/#G)) scores were relatively constant along the HIV-1 genome. Red shaded circles – HM3G sequences; blue open circles – NH sequences; nucleotide position relative to HXB2 (GenBank Accession Number K03455); Y-axis values are log 10 transformed; curves are locally weighted (Loess) smoothers).
Vif peptide sequences derived from the HM-3G group were significantly different
from NH vif sequences in terms of a higher overall rate of non-consensus amino
60 acids (Figure 1.28, Mantel-Haenszel, P<0.0005). The strongest associations were tryptophan to in-frame stop substitutions at positions 70, 89, 174 and 21 and substitution of isoleucine for the methionine start codon (all Pc<0.04, Bonferroni).
These substitutions, which would be anticipated to encode for truncated and functionally defective vif proteins, are all in the trinucleotide sequence context targeted by APOBEC3G (TGG) and therefore are likely to have resulted from, rather than caused HIV-1 hypermutation. HM-3G was also associated with an R90K polymorphism (Pc=0.07, Bonferroni); again however, this could be attributed to the action of APOBEC3G. No other vif polymorphisms were found to be significantly associated with APOBEC3G-associated hypermutation (all Pc>0.4).
Regarding the potential role of truncated vif sequences in permitting APOBEC3G- mediated HIV-1 hypermutation, it is notable that while HM-3G was significantly associated with in-frame stop codons specifically in vif (P<0.001), in-frame stop codons in non-vif or non-vif , non-env proteins were not associated with HM-3G sequences (P=0.20 and P=0.63, respectively). Additionally, HIV-1 hypermutation was not evident in the two sequences in which the only vif in-frame stop codon was at vif amino acid positions 153 (leucine) or 174 (tryptophan). In these sequences,
G→A substitutions accounted for only 20.7% and 24.9% of all substitutions, affecting only 4.0% and 5.0% of consensus guanine residues, respectively. It has previously been demonstrated that a naturally occurring vif protein truncated at tryptophan 174 is indistinguishable from wild type vif in maintaining viral infectivity 73 .
61 80
70
60 motif required for β- sheet formation 1 50
40
30 motif required for β- sheet formation 1
20
10
0 MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQ 10 20 30 40 50 60 70 80 90 80
70 Vif motif required for multimerisation and functionality 3
60
50 SOCS-box 40 N-terminal C-terminus 19 amino acids not polymorphismpolymorphism frequencyfrequency(%) (%) BC-box required for vif function 4 2 30 motif
20 10
0 VDPDLADQLIH LYYFDC FSESAIRNAILGHRVSPRC EYQAGH NKVGSLQYLALAALITPKKIKPPLPSVTKLTEDRWNKPQKTKGHRGSHTMN GH* 100 110 120 130 140 150 160 170 180 190 NH consensus vif amino acid sequence
Figure 1.28 Consensus vif amino acid sequence and polymorphism frequency among non-hypermutated sequences. The population consensus sequence shown here was identical to the consensus vif sequence of hypermutated HIV-1 sequences. Numbers indicate vif amino acid number; * - stop codon; 1 – reference 134; 2 – reference 47; 3 – reference 92; 4 – reference 73.
62 Here, the occurrence of a naturally occurring vif protein truncated at leucine 153 also suggests that the C-terminal 39 amino acids are dispensable for this protein’s roles in promoting viral infectivity as well as counteracting APOBEC3G. Interestingly, despite the codon for tryptophan being a target for APOBEC3G, tryptophan residues
5, 11 and 79 were entirely conserved in the hypermutated vif sequences and are therefore unlikely to play a role in inhibiting APOBEC3G or APOBEC3F activity, in contrast to data from Tian and colleagues 124 .
Figure 1.28 demonstrates the high degree of vif polymorphism that appears to be tolerated without apparently increasing viral susceptibility to the effects of
APOBEC3G and/or APOBEC3F. Within the non-hypermutated group, polymorphism was observed at the serine 144 and leucine 148 residues of the SOCS-box
N-terminal BC-box motif ( 144 SLQYLA149 ) required for vif phosphorylation 103 and
Elongin BC and Cul5 binding 47 , respectively, and the proline 162 (1.0%) residue of the
SOCS-box C-terminal motif 161 PPLP 164 required for vif multimerisation and subsequently vif function 92 . In contrast, the existence of polymorphism at these sites in our population suggests that they may not be essential for maintaining vif
125 function. However, the recently described H x5 Cx17-18 Cx3-5H zinc-binding motif , critical for assembly and activity of the vif -Cul5-E3 ligase 126 , was entirely conserved.
We also identified vif polymorphisms that were unique to the HM-3G sequences within this study population, given previous evidence that sequence variation at a number of critical residues can affect vif -APOBEC3G interactions 50,127 . Twenty- three vif amino acid variants were found uniquely in HM-3G sequences (Figure
1.29), of which 82% were within regions previously shown to be required for vif -
63
I K15 * * K * E K S K*K K* K HM-3G M1 M8 W21 M29 W38 E45 W7 G75 R77 G82 E88 W89 R90 R173 W174 G185 W193
* HM-3G W174 W193
* * * P HM-3G M1 W21 W70 W89 H127 W193
K IK K* D K K E NK * S HM-3G M1E2 M16 E45 E88 W89 R92 N101 E134 G138 D172 R173 W174 G182 W193
K * E K HM-3G M1E2 W70 K91 S95 W193
I * V K HM-3G 1 8 70 74 90 193 M M W T R W
I A * I * HM-3G M1 M8 E54 W70 A123 W174 W193
K HM-3G R77 W193
HM-3G M1 W193
* * * K HM-3G M1 W21 M29 W70 W89 R90 W193
L S HM-3F M1 V98 A103 W193
* NH M1 W174 W193
* NH M1 L153 W193
Figure 1.29 Schematic representation of HM and truncated vif proteins identified in vivo from putative translation of proviral DNA sequences. HM-3G – APOBEC3G-mediated hypermutated sequence; HM-3F – APOBEC3F-mediated hypermutated sequence NH – truncated vif proteins translated from non-hypermutated proviral sequences; shaded bar indicates translated vif sequence; unshaded bar indicates untranslated vif sequence; partially shaded bar indicates region not sequenced; red line indicates unique polymorphism from consensus that could not potentially be attributed to the corresponding APOBEC3 enzyme; blue line indicates unique polymorphism from consensus that could potentially be attributed to the corresponding APOBEC3 enzyme; sequences are ordered by HM-3G, HM-3F and NH in descending consolidated 3G score; amino acid(s) below the peptide indicate consensus, amino acids above the peptide indicate amino acid(s) unique to the individual peptide sequence.
64 APOBEC3G interaction 50,127 . Of these, thirteen could not have resulted from an
APOBEC3G-mediated G →A substitution of the NH consensus amino acid, all of which were located within the vif N-terminus. It is also interesting to note that a charged consensus amino acid was present in 14/23 of these positions, and that the variant vif sequence was frequently associated with either a reversal of the charge state (6/14,43%) or substitution of a neutral amino acid (3/14, 21%). An additional three vif amino acids were unique to the two HM-3F vif sequences available and could also not have resulted from a G →A substitution of any of the alternative amino acids present in the NH sequences. Again, two of the HM-3F unique amino acids were located within the vif N-terminus (L 98 , S 103 ).
1.3.6 APOBEC3G GENETIC VARIATION AND G→→→A
HYPERMUTATION
To assess the contribution of APOBEC3G genetic variation to hypermutation, we sequenced the entire 10.5 kb APOBEC3G gene, including 2 kb 5’ of the transcription start site and 0.5 kb 3’ of the 3’UTR. We initially compared the frequency of APOBEC3G alleles from 8 patients with the greatest G →A burden to that estimated from a pooled DNA sample (187 Caucasian individuals 118 ) as control, based on relative chromatogram peak height. As demonstrated in Table 1.2, the
APOBEC3G amino acid sequence was highly conserved in this predominantly
Caucasian study population. Based on the estimated allele frequencies of the 22 single nucleotide polymorphisms (SNPs) identified (17 previously described and 5 novel SNPs) and the sample size, we had sufficient power (Supplementary Figure
1.32S) to detect significant differences in allele frequencies between the patients
65 with hypermutated sequences for SNP positions 625 (rs9607609) and 6,892
(rs5757467) relative to the transcription start site (Table 1.2). We then genotyped the
625 and 6,892 SNPs for patients that had DNA available (n=119). The frequency of the 6,892 C allele tended to be higher in patients with evidence of APOBEC3G- mediated hypermutation (50.0% versus 29.9%, P=0.062), with homozygosity for the
6,892 C allele present in 25% of patients with evidence of APOBEC3G-mediated hypermutation compared with 7.5% of non-hypermutated sequences (P=0.082).
Table 1.2 APOBEC3G allele frequencies among 8 patients with marked G →→→A hypermutation. Comparisons with a pooled DNA control sample and a published study (Do et al ,110 ).
Control Do et al , HM-3G NCBI 2005 dbSNP ID Position Allele Allele Allele 2 Allele 1 Allele A1.A1 A1.A2 A2.A.2 1 2 2 7291971 -1,193 0.60 0.40 0.40 0.44 0.56 2 3 3 6519166 -1,190 0.70 0.30 0.31 0.56 0.44 2 5 1 5750743 -321 0.50 0.50 0.28 0.44 0.56 1 5 2 12160242 480 >0.90 <0.10 0.09 0.94 0.06 7 1 0 9607609 625 0.80 0.20 ND 0.38 0.62 2 2 4 5757465 3,756† 0.60 0.40 0.51 0.75 0.25 4 4 0 12158985 5,985 >0.90 <0.10 0.07 0.88 0.12 6 2 0 2294366 5,986 0.70 0.30 0.34 0.56 0.44 2 5 1 2294367 6,207 0.60 0.40 0.42 0.44 0.56 2 3 3 nl 6,848 >0.90 <0.10 ND 0.88 0.12 6 2 0 3091374 6,878 0.50 0.50 ND 0.44 0.56 2 3 3 5757467 6,892 0.70 0.30 ND 0.31 0.69 0 5 3 5757468 6,893 0.70 0.30 ND 0.56 0.44 2 5 1 nl 6,894 >0.90 <0.10 ND 0.88 0.12 6 2 0 nl 8,984‡ >0.90 <0.10 ND 0.88 0.12 6 2 0 5757471 9,218 >0.90 <0.10 ND 0.94 0.06 7 1 0 28474760 9,250 >0.90 <0.10 0.07 0.88 0.12 6 2 0 28474761 9,432 >0.90 <0.10 0.12 0.88 0.12 6 2 0 nl 10,393 0.50 0.50 ND 0.92 0.08 5 1 0 nl 10,399 0.70 0.30 ND 1.00 0.0 8 0 0 5757472 10,600 0.50 0.50 ND 0.44 0.56 1 5 2 3891126 10,811 >0.90 <0.10 ND 0.88 0.12 6 2 0 Allele frequencies of the pooled DNA sample were estimated by chromatogram relative peak height. Significant differences in the allele frequencies of the 625 and 6,892 SNPs were evident between the hypermutated patients and the pooled DNA sample. NCBI dbSNP ID – National Centre for Biotechnology Information SNP database reference number; † - synonymous SNP; ‡ - non- synonymous SNP; A1.A1 – number of patients homozygous for allele 1; A1.A2 – number of heterozygous patients; A2.A2 – number of patients homozygous for allele 2; <0.10 – allele frequency below the limit of detection; nl – novel SNP; ND – not determined; positions are relative to the transcription start site with reference to NCBI accession number NT_011520.
66 However, this marginally significant univariate association was abrogated after adjusting for multiple comparisons (Pc>0.5). The frequency of the 625 C allele was similar amongst patients with evidence of APOBEC3G-mediated hypermutation and patients harbouring NH sequences (allele frequency, 75.0% versus 77.2%, P=0.80;
CC genotype frequency, 58.3% versus 57.3%, P=1.0).
1.3.7 G→→→A HYPERMUTATION AND HIV-1 VIREMIA
The biological and clinical relevance of HIV-1 hypermutation in proviral DNA sequences is ultimately a function of its impact on productive HIV infection, measured by the level of viremia in plasma samples. We therefore sought to investigate the effect of HIV-1 hypermutation on pre-treatment viremia in this study population. Univariate analysis indicates that patients harbouring hypermutated sequence as defined had significantly lower viral load than those with non- hypermutated sequence (4.32 ± 0.60 versus 4.98 ± 0.75 log 10 copies HIV RNA/mL,
P=0.001). Utilising linear regression models, this association between hypermutation and lower pre-treatment viremia remained highly significant (P=0.013) even after adjusting for CD4 + percent (P<0.001) and presence of at least one of the known protective host alleles, CCR5 ∆32 128 , HLA-B57, HLA-B58 or HLA-B27 129 (P=0.096) and approximates 67% and 40% reductions in viral loads attributable to hypermutation and host protective alleles, respectively. Hence, the clinical influence of HIV-1 hypermutation appears to be demonstrable within this group of patients who manifest a marked preference for G →A substitutions in an APOBEC3G or
APOBEC3F sequence context.
67 1.4 DISCUSSION
The data presented in this population-based study of clade B HIV-1 sequences suggest that HIV-1 G →A hypermutation is a prevalent phenomenon associated with
significantly reduced plasma HIV RNA levels in vivo , indicating that APOBEC3G
and 3F-mediated hypermutation can take its place alongside other protective host
genetic factors as a clinically and biologically relevant antiretroviral restriction
factor. Indeed, the reduced HIV-1 viremia associated with hypermutation was
substantially greater than that exerted by known host factors such as the CCR5 ∆32 chemokine receptor variant and remained highly significant after adjusting for these variables as well as for the influence of CD4 + count. Such reductions in viral load are substantially greater than those previously associated with protective HLA alleles 130 and are comparable to the effects of zidovudine monotherapy in early studies of antiretroviral treatment 131 .
Here, hypermutated proviral DNA was estimated in 12% of study participants, utilising a bulk PCR sequencing approach that approximates the dominant species within a heterogeneous viral population. In many respects these findings complement the work of Kieffer and colleagues 132 who examined the phenomenon of in vivo hypermutation in detail utilising 319 pol clones derived from 9 patient samples. This study revealed at least one hypermutated proviral DNA sequence from each individual examined, although hypermutated sequences accounted for less than
10% of the proviral population within an individual. Similar results have been obtained by Janini et al 19 who detected the presence of G →A hypermutation in 45% of HIV protease sequences from a Tanzanian study population. These observations
68 are consistent with in vitro data demonstrating low-level APOBEC3G and 3F- mediated cytidine deaminase activity directed against HIV-1 sequences even in the presence of functional vif 75,108 . It has also been suggested that the rapid turnover of vif through proteasomal degradation contributes to constitutively low cellular protein expression 133 , thereby providing an incomplete barrier to APOBEC3G and/or 3F activity. In this study, we now provide evidence that hypermutated proviral HIV-1
DNA sequences can be demonstrated by bulk PCR methods in a significant minority of clade B HIV-1-infected individuals. Taken together, these data suggest that
APOBEC3-mediated cytidine deamination is highly prevalent but effectively constrained by functional interactions with vif , so that G →A hypermutation is generally confined to a minor proportion of the overall viral population.
It should be noted that the sequence context targeted by APOBEC3G appears to specifically select for loss-of-function mutations, including within the vif genomic sequence. For example, targeted substitutions involving tryptophan residues (T GG) produce in-frame stop codons (T AG) while the vif start codon and adjacent aspartic acid residue also create an APOBEC3G target motif (AT GG) that results in loss of the methionine initiation signal following G →A substitution. To some extent this susceptibility appears to have been overcome by the utilisation of multiple alternative start codons at methionine residues 8, 16 and 29, although it is notable that the capacity to counteract APOBEC3G activity appears to be lost as a consequence of these N-terminal truncations 123,133 . In this study, substitutions associated with truncated vif sequences could be entirely explained by APOBEC3G- mediated cytidine deamination (Figure 1.29), although it is notable that hypermutation was associated specifically with vif in-frame stop codons but not with
69 in-frame stop codons in non-vif viral peptides. Hence, while it is difficult to attribute
causality in the relationship between hypermutation and defective vif , the most
parsimonious explanation appears to be provided by a mechanism in which
APOBEC3-mediated G →A substitutions, occurring in a relatively non-permissive
cellular environment but targeting T GG trinucleotide motifs stochastically along the
genome, can become unconstrained if this process selects for defective vif sequences.
Although the translated vif sequences in this cohort were highly polymorphic (Figure
1.28), there were 23 vif amino acid variants unique to HIV-1 hypermutated
sequences, of which 13 associated with HM-3G sequences and three unique to HM-
3F sequences could not have resulted from APOBEC3G-mediated cytidine
deamination (Figure 1.29). These data are consistent with those that suggest the vif
N-terminal contains distinct motifs that are likely to interface with APOBEC3G and
APOBEC3F 123,124,127 , and may therefore represent true vif allelic variants that facilitate hypermutation. Although the associated amino acid differed, four of the twenty-three vif amino acids unique to HM-3G sequences (K 45 , E 75 , E 138 , K 185 ) occurred at positions previously associated with naturally occurring non-functional vif variants 123 .
With reference to the non-hypermutated vif sequences, we observed polymorphism
at residues within key motifs shown to be required for vif phosphorylation 103 , vif -E3 ligase complex formation 47 , vif multimerisation 92 and β-sheet formation 134 . In this
study however, these variants were not associated with APOBEC3-mediated
hypermutation, suggesting that polymorphisms within these motifs are tolerated
without significantly compromising vif function. While it is unclear if these
70 polymorphisms alter the efficiency of polyubiquitination and proteasomal degradation of the vif -APOBEC3G-E3 ligase complex in vivo , the lack of hypermutation in these sequences could potentially be attributed to an undiminished capacity for vif -APOBEC3G complex formation, which is sufficient to restrict
APOBEC3G-mediated hypermutation in vitro 108 . Despite the high degree of vif polymorphism, several stretches of conserved residues, most notably the first 18 N- terminal amino acids, were observed that may represent novel drug targets.
The dinucleotide sequence context of the G →A substitutions suggests that
APOBEC3G was the dominant contributor to hypermutation in this study group.
These findings are consistent with the previously mentioned in vivo study by Kieffer et al 132 , involving clade B HIV-1 sequences. In contrast, two studies involving
Tanzanian study populations infected with predominantly non-clade B virus 19,20 indicate less bias towards the APOBEC3G context. These differences warrant further exploration, particularly as a non-synonymous APOBEC3G 186R polymorphism associated with accelerated progression to AIDS (and therefore implying loss of
APOBEC3G function) has been found to be frequent in African Americans (37%) but rare in European Americans (5%) 109 . Hence, a relatively greater contribution of
APOBEC3F to HIV-1 cytidine deaminase editing may be anticipated in at least some populations of African origin. In this study, there was no convincing evidence that
APOBEC3G genetic variation contributed to the development of HIV-1 hypermutation, and it is notable that we were unable to identify significant amino acid variation within APOBEC3G sequences in these Caucasian populations (Table
1.2). A marginal univariate association between an intronic APOBEC3G 6,892 C allele and hypermutation was demonstrated, although the role of this intronic
71 polymorphism on APOBEC3G function is unclear. It is however possible that functional polymorphism are located in the as-yet-undefined promoter or regulatory regions of this genetic locus, and in this regard it is notable that APOBEC3G mRNA levels in peripheral blood mononuclear cells have recently been found to exhibit an inverse correlation with HIV-1 pre-treatment viremia 135 .
In conclusion, this study provides strong support for the proposition that
APOBEC3G-mediated cytosine deamination provides a potent source of innate HIV-
1 restriction at the population level. Moreover, the prevalence and antiretroviral
impact of APOBEC3G and APOBEC3F-mediated HIV-1 hypermutation within the
study population can be compared favourably with known host protective factors.
Thus, less than four years after Malim and colleagues first isolated the APOBEC3G
gene and tentatively proposed a cytosine deaminase function for its product 11 , we
submit that these data fulfil the promise contained within these early in vitro studies, that APOBEC3G comprises a form of innate antiviral resistance that is clinically and biologically relevant and which may lend a fresh perspective to the area of
HIV/AIDS therapies 18 .
72 1.5 SUPPLEMENTARY DATA
Table 1.3S Oligonucleotide sequences used for the amplification and sequencing of the entire human APOBEC3G gene, including 2.0 kb 5’ of the transcription start site and 1 kb 3’ of the transcription stop site.
Oligo Name Sequence (5’- 3’) 5’ Binding Position 154F AGACCTGGCAAATTTTGGTG 18,861,852 6636R GGCCTCTCACTCACAGGAAG 18,868,334 5117F GTGATGCTTCAACAGCAGGA 18,866,874 13356R GTGTCTAGGCCAGGGAAACA 18,875,054 902F TTTTTGGCTGTTCCAGACTT 18,862,600 1581F ACATCTGAGGCCCACACC 18,863,279 2107F AAAACCAGAAGCTTGGAGCA 18,863,805 2893F CCAGCATGGATAAACCCTGT 18,864,591 3556F CGGAATTTCGCTCTTGTCG 18,865,254 4172F TCTTGTTGCTCAGGCTGGA 18,865,870 4899F TCAGGTGCCCAGGTCTCTAA 18,866,597 5647F TGGCAAGATCTCCTGGACTC 18,867,345 6442F CTCTCACCTCCTGCTCATCC 18,868,140 7109F GCCCACACTGAAGGTCTCAT 18,868,807 7852F CCAGAATCTTCCCAGTTCCA 18,869,550 8595F TGAACCTTGGGTCAGAGGAC 18,870,293 9039F GCCAAGTGGGATCAAAGAAA 18,870,737 9449F GGCCTACCTGACCTCACAAG 18,871,147 10191F AGGGGAATTGACGGGTAGAC 18,871,889 10931F AGCCTGAACCACATGGAGAA 18,872,629 11717F AGGGAAAGGAGGGACTGAAA 18,873,415 12489F CAAACCTACTAATCCAGCGACA 18,874,187 930R GCAGCCAAGAAGTCTGGAAC 18,862,628 1617R GAGGGAGGGACAGCAGGT 18,863,315 2373R GACAACACAGCAGGGCAAG 18,864,071 3193R GGACCGGGCTCTCTTCTTAC 18,864,891 3630R GAATTTCGGCGGAGACTGTA 18,865,328 4276R TCGCAGCTACTTGCGCGTCT 18,865,974 4960R ACATGGCCAAGGGACTACTG 18,866,658 5813R CACTTGCTGAACCAGTGGAA 18,867,511 7162R GGAGACAGGTCCTTTCATCAG 18,868,860 7908R GGATGCCACCTTTGATAGGA 18,869,606 8713R TGGTCACCTGGTTGCATAGA 18,870,411 9556R TCAGGACAGAATCCCTGGTC 18,871,254 10263R GGAGGACAGATGCCACTGAG 18,871,961 11095R AAAGAAAGCAAGCCATGAGG 18,872,793 11738R GGTTTCAGTCCCTCCTTTCC 18,873,436 12519R GATTCAAATTGTCGCTGGATT 18,874,217
5’ binding position relative to NCBI accession number NT_011520.
73
Exon 1 Exon 2 Exons 3 & 4 Exon 5 Exons 6, 7 & 8 Introns 3 & 4 Introns 5 & 6 Intron 7
Promoter ?? UTR Intron 1 Intron 2
154 5,177 6,636 13,356
6,442 7,162 7,852 8,713 154 930 1,581 2,373 7,109 7,908 8,595 9,556
902 1,617 2,107 3,193 9, 449 10,263 10,931 11,738 12,489 13,356 2,893 3,630 4,172 4,960 5,647 6,636 10,191 11,095 11,717 12,519 3,556 4,276 4,899 5,813
Figure 1.30S Binding sites of oligonucleotide primers used for the amplification (black) and sequencing (blue and red) of the entire human APOBEC3G gene, including 2kb upstream of the transcription binding site and 0.5kb 3’ of the 3’UTR. Numbers represent nucleotide binding site of 5’ end of the oligonucleotide primer on reference sequence NCBI accession number NT_011520.
74
95:5
90:10
85:15
80:20
75
75:25
70:30
60:40
50:50
Figure 1.31S Chromatograph results of sequencing pooled PCR products homozygous for alternative alleles at varying proportions to estimate the lower limit of allele detection by sequencing. Ratios represent the ratio at which PCR products homozygous for the T allele were mixed with PCR products homozygous for the G allele prior to sequencing.
76
Power vs P1 by N1 with P2=0.38 Alpha=0.05 N2=16 Power vs P1 by N1 with P2=0.38 Alpha=0.05 N2=42 2-Sided Prop Test 2-Sided Prop Test 1.0 1.0
0.8 0.8 50 50 0.6 0.6 100 100
N1 N1
Power 200 Power 200 0.4 0.4 300 300 400 400 0.2 0.2
0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 P1 P1
Power vs P1 by N1 with P2=0.44 Alpha=0.05 N2=16 Power vs P1 by N1 with P2=0.44 Alpha=0.05 N2=42 2-Sided Prop Test 2-Sided Prop Test
1.0 1.0
0.8 0.8
50 50 0.6 0.6 100 100 N1 N1
Power 200 Power 200 0.4 0.4 300 300 400 400 0.2 0.2
0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 P1 P1
Power vs P1 by N1 with P2=0.56 Alpha=0.05 N2=16 Power vs P1 by N1 with P2=0.56 Alpha=0.05 N2=42 2-Sided Prop Test 2-Sided Prop Test 1.0 1.0
0.8 0.8
50 50 0.6 0.6 100 100 N1 N1 Power 200 Power 200 0.4 0.4 300 300 400 400 0.2 0.2
0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 P1 P1
77
Power vs P1 by N1 with P2=0.69 Alpha=0.05 N2=16 Power vs P1 by N1 with P2=0.69 Alpha=0.05 N2=42 2-Sided Prop Test 2-Sided Prop Test 1.0 1.0
0.8 0.8
50 50 0.6 0.6 100 100 N1 N1
Power 200 Power 200 0.4 0.4 300 300 400 400 0.2 0.2
0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 P1 P1
Power vs P1 by N1 with P2=0.88 Alpha=0.05 N2=16 Power vs P1 by N1 with P2=0.88 Alpha=0.05 N2=42 2-Sided Prop Test 2-Sided Prop Test 1.0 1.0
0.8 0.8
50 50 0.6 0.6 100 100 N1 N1
Power 200 Power 200 0.4 0.4 300 300 400 400 0.2 0.2
0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 P1 P1
Power vs P1 by N1 with P2=0.94 Alpha=0.05 N2=16 Power vs P1 by N1 with P2=0.94 Alpha=0.05 N2=42 2-Sided Prop Test 2-Sided Prop Test 1.0 1.0
0.8 0.8
50 50 0.6 0.6 100 100 N1 N1
Power 200 Power 200 0.4 0.4 300 300 400 400 0.2 0.2
0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 P1 P1
Figure 1.32S Power calculations used to determine sample size required to detect significant differences in APOBEC3G allele frequencies between HM-3G and NH sequences based on the allele frequencies of the HM-3G sequences and those estimated from a pooled DNA control sample.
78
CHAPTER TWO
2 CHARACTERISATION OF THE HUMAN
DEOXYTHYMIDYLATE KINASE AND THYMIDINE KINASE 2
GENES AND THEIR INFLUENCE ON AZT AND D4T
TOXICITIES
79 2.1 GENERAL INTRODUCTION AND REVIEW OF THE LITERATURE
2.1.1 AZT AND D4T THERAPY
Zidovudine (AZT; ZDV; 3’-azido-3’-deoxythymidine; Retrovir™) and stavudine
(d4T; 2’,3’-didehydro-3’-dideoxythymidine; Zerit) are currently recommended components of highly active antiretroviral therapy (HAART) for the treatment of
HIV-1 infection in adults, adolescents and children by the US Department of Health and Human Services ( http://aidsinfo.nih.gov ). Initially intended to treat cancer, AZT was first synthesised in 1964 under a US National Institutes of Health grant, but failed to show efficacy and had an unacceptably high side effect profile. Then in
1985, through collaborations between scientists at the National Cancer Institute
(NCI) and Burroughs Wellcome Co., AZT proved effective in vitro against HIV and was clinically demonstrated to increase CD4+ counts in AIDS patients leading to
Federal Drug Administration (FDA) approval for use against HIV, AIDS, and AIDS
Related Complex on March 20, 1987, and then as a preventive treatment in 1990;
AZT was the first compound approved by the FDA for the treatment of HIV-1
infection 136 .
Stavudine, the fourth drug approved by the FDA for the treatment of HIV-1 infection, was initially approved for the treatment of patients with advanced HIV disease intolerant to, or experiencing progression of disease on, previously approved drugs. Other NRTIs currently approved for treatment of HIV-1 infection include zalcitabine (ddC; 2’,3’-dideoxycytidine; HIVID), didanosine (ddI; 2’,3’- dideoxyinosine; VIDEX), lamivudine (3TC; 3’-thiacytidine; cis -1-[2’-
hydroxymethyl-5’-(1,3-oxathiolanyl)]cytosine; EPIVIR), emtricitabine [(-)FTC],
80 abacavir (ABC; 4-(2-amino-6-cyclopropylamino-9H-purin-9-yl)-1-cyclopent-2- enyl]methanol; Ziagen) and tenofovir (TDF; Viread) (TDF is a nucleotide analogue rather than a nucleoside analogue)137 .
Both AZT and d4T are highly effective inhibitors of viral replication 138-144 and as such are integral components of contemporary HIV-1 treatment regimens 141 .
However, their utilisation is limited by their toxicity. This review will address AZT and d4T pharmacology, the mechanisms of their toxicities and the role of pharmacogenetics in HIV-1 therapy.
2.1.1.1 MODE OF ACTION OF NRTI S
NRTIs have a wider spectrum of antiviral activity than the other classes of HIV-1 therapies; protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) 145 . As illustrated in Figure 2.1, NRTIs are synthetic analogues of naturally occurring nucleosides. The 5’-triphosphate metabolite of NRTIs differ from those of naturally occurring nucleosides in that they lack the hydroxyl group at the 3’-position of the ribose ring that acts as an acceptor for the covalent attachment of subsequent nucleosides 5’-monophosphate 145-147 .
NRTIs inhibit HIV-1 replication by two mechanisms. Firstly, as their name implies,
NRTIs inhibit HIV-1 reverse transcriptase (RT), the enzyme required to catalyse the synthesis of proviral double-stranded DNA from the RNA template, by binding to the RT active site. Secondly, NRTIs may become incorporated into the nascent DNA
81 chain in place of naturally occurring endogenous nucleotides resulting in termination of DNA elongation 145 .
Figure 2.1 Structure of nucleoside reverse transcriptase inhibitors and their corresponding deoxynucleoside. ADV and TDF are nucleotide analogues of adenosine and are shown with their corresponding nucleotide. Taken from reference 148 (reprinted by permission from Bentham Science Publishers Ltd) [ Curr. Med. Chem. – Anti-Infective Agents , 2003;2:241-62].
82 2.1.2 CLINICAL TOXICITIES OF THYMIDINE -ANALOGUE THERAPY
Clinical toxicities associated with NRTI therapy are tissue-specific manifestations of mitochondrial toxicity and resemble those seen in inherited mitochondrial diseases 148,149 and mitochondrial abnormalities are typically observed histologically in NRTI-associated myopathy, lipoatrophy and neuropathy. Toxicities mediated by mtDNA depletion and subsequent deficiencies in oxidative phosphorylation are commonly observed with both AZT and d4T, but more commonly with d4T therapy, due in part, to the greater potency of d4T to inhibit mitochondrial DNA polymerase γ compared to AZT 150,151 , and in part, to tissue differences in expression of cellular kinases required for their anabolism152 . For example, lactic acidosis, hyperlactataemia 153-159 , lipoatrophy 148,160-168 , pancreatitis 160 and neuropathy 161-163 are more commonly observed in d4T recipients than AZT recipients. However, AZT induces mitochondrial toxicity independent of mtDNA depletion and is therefore associated with certain toxicities more commonly than d4T therapy. These include myopathy 164-166 and haematological toxicities such as anaemia 167-169 and neutropenia 170 . The mechanisms of toxicity shall be discussed in sections 2.1.3 and
2.1.4.
2.1.2.1 HAEMATOLOGICAL TOXICITIES
Haematological toxicities are the most common toxicities observed with AZT therapy but rarely associated with d4T therapy 167-171 . In early clinical trials of AZT in patients with advanced AIDS disease using doses of 1500mg/day, Grade 3 marrow suppression was evident in 45% of AZT recipients; anaemia (Hb <7.5g/dL) and
83 neutropenia was observed in 24% and 16% of AZT recipients, respectively, compared to only 4% and 2% of HIV-1+ antiretroviral therapy naïve patients.
Furthermore, 31% of AZT recipients compared to only 11% of placebo recipients required red-cell transfusions (P<0.05). Such prevalence of toxicity led to reductions in AZT doses to 500mg/day which was demonstrated to be as efficacious as
1500mg/day regimens in delaying progression to AIDS, increasing CD4 + counts and
reducing p24 antigen, but significantly reduced the frequency of haematological
toxicities from 11.8% to 2.9% 172,173 . Even in the HAART era, haematological toxicities remain common. From studies of contemporary HAART regimens, a meta- analysis of six prospective, randomised, comparative studies conducted by Moyle et al 167 , all grades of anemia and neutropenia were more frequently observed with AZT therapy (5%-18% and 26%-43%, respectively) than d4T therapy (4%-9% and 15-
31%, respectively).
Despite the substantial reductions in the frequency of AZT-associated haematological toxicities owing to dose-reductions, such toxicities remain clinically relevant due to their influence on disease progression 174-176 . In the large,
observational, prospective EuroSIDA study including 6,725 HIV-1+ patients, 40.8%
and 15.9% of patients with severe and moderate anaemia at baseline respectively,
died within 12 months of recruitment compared to only 3.1% of patients with mild
anaemia. In a multivariate time-updated Cox proportional hazards model, as little as
1g/dL decrease in the latest haemoglobin level increased the hazard of death by 57%
and had a greater effect than a 50% drop in CD4 + count (50% drop) or a log decrease
in viral load (37% drop) 174 .
84 Macrocytosis develops in most AZT and d4T recipients within weeks, albeit less severe in d4T recipients than AZT recipients 177-182 . Macrocytosis is not however, a marker of haematological toxicity; it can occur in the absence of haematological toxicity 183 and in fact, elevated mean corpuscular volume (MCV) can actually define the subgroup of patients resistant to AZT-associated erythroid hypoplasia and consequent anemia 184 , and is a useful marker of adherence to thymidine-analogue therapy 185 .
2.1.2.2 MYOPATHY
NRTI-associated myopathies are almost exclusively observed with AZT therapy, occurring in at least 17% of patients in the HAART era and in up to 33% in the pre-
HAART era 186-188 . Symptoms of myopathy, including fatigue, muscle weakness, and elevation of muscle enzymes (creatine kinase or aldolase in some cases) often begin after the first 6 months of therapy and are often reversible, clinically and histologically, within as little as 3 months following discontinuation of
AZT 164,165,187,189-191 . Clinically, AZT myopathy mimics HIV-associated polymyositis to such an extent that debate exists as to whether they represent one disease or two 190,192 . However, it has been reported by several groups that AZT myopathy can be distinguished from HIV-associated polymyositis by the presence of “AZT fibres”, a termed coined by Dalakas and colleagues 193 to designate atrophic ragged red fibres with marked myofibrillar alterations, including thick myofilament loss and cytoplasmic body formation 194-196 , although they have also been observed in HIV-
1+, AZT-naïve individuals 191 . Mitochondrial dysfunction is common to both AZT- associated myopathy and HIV-associated polymyositis191,197 , however impaired
85 cytochrome c oxidase (complex IV of the mitochondrial respiratory chain comprised of nDNA and mtDNA encoded subunits) activity is a unique and consistent feature of AZT-associated myopathic fibres, even in the absence of histological changes 194
(Figure 2.2).
AB
Figure 2.2 Focal cytochrome oxidase deficiency is a unique and consistent feature of AZT- associated myopathy. (A) AZT-associated myopathy is characterised by focal cytochrome oxidase deficiency. Cryostat section, x450 magnification before 8% reduction. (B) HIV-associated myopathy characterised by normal cytochrome oxidase deficiency. Cryostat section, x280 magnification before 8% reduction. Taken from reference 194 (reprinted by permission from Bentham Science Publishers Ltd) [ Acta Neuropathol (Berl) , 1993;85:431-6].
2.1.2.3 LACTIC ACIDOSIS AND HYPERLACTATAEMIA
Lactic acidosis (LA) and hyperlactataemia (HL) are probably the most recognisable
features of mitochondrial dysfunction in clinical disease and represent varying
degrees of derangement in systemic homeostasis 198-200 . At one end of the spectrum,
LA indicates an extreme ‘decompensated’ metabolic state in which homeostasis is completely lost, while HL represents the other end of the spectrum, a ‘compensated’ homeostatic system in which elevated lactate production is balanced by effective mechanisms of lactate clearance 156,198 .
86 Lactate is produced in the cytoplasm during the glycolytic pathway as a product of trans-formation of pyruvate and is primarily metabolised oxidatively in mitochondria, with no intracellular accumulation of lactate. However, in the absence of oxygen or a functional respiratory chain, lactate can accumulate in the cytoplasm and result in increased lactate production 200 .
HL may be symptomatic or asymptomatic. Asymptomatic HL (generally <2.5 mmol/L) is a benign syndrome and affects between 9 and 42% of antiretroviral recipients and approximately 2% of antiretroviral therapy-naïve outpatients 156,201-206 .
In the WA HIV Cohort Study, chronic mild asymptomatic HL (lactate concentrations
1.5-3mmol/L), maintained over many months, was the most common pattern of HL observed, indicating that overall, lactate concentrations rise from the pre-treatment baseline in most NRTI-recipients but are largely maintained at only mildly elevated levels in the long term 156 (Figure 2.3). Similar observations have been reported from the Swiss HIV-1 Cohort Study by Imhof et al 201 and such mild elevations are not considered to warrant therapy revision 156,205 .
Symptomatic HL is characterised by mild to moderate increases in plasma lactate concentration (<2.5 mmol/L), non-specific symptoms including nausea, vomiting and abdominal pain, in the absence of metabolic acidosis or hepatic steatosis.
Reported prevalence of clinically relevant HL is approximately 1-3% 201,207 and is predominantly associated with d4T and ddI therapy 200,201,204,208 . From the French
HIV-1 Cohort Study, substitution of d4T and/or ddI in hyperlactataemic patients decreased HL prevalence from 25% to 3% 204 and Garcia-Benayas et al 209 reported significant reductions in plasma lactate concentrations in patients that substituted
87 abacavir for d4T compared to those that continued d4T therapy. Large observational studies have also identified associations with the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz and boosted- and double-protease inhibitor regimens 201,210,211 . Host-related risk factors for development of HL include older age, increased waist-hip ratio, high CD4 + cell count and liver dysfunction 201 .
Figure 2.3 Rise in population average venous lactate concentrations after start of highly active antiretroviral therapy (HAART) in patients treated with stavudine (dashed line) and zidovudine (solid line). Taken from reference 156 (reprinted by permission from Lippincott Williams & Wilkins) [AIDS , 2001;15:717-23].
LA, characterised by persistently elevated plasma lactate levels (usually >5mmol/L), metabolic acidosis (pH <7.35 or bicarbonate <20mmol/L) and almost always accompanied by hepatic steatosis, is an uncommon but life-threatening complication of antiretroviral therapy 156 . LA is unpredictable in that it can occur rapidly in patients who have been on stable NRTI therapy for months or even years and is poorly predicted by HL 156 . Reported incidence rates range from 1.3 to 10 cases per 1000 person years of antiretroviral therapy depending on the definition 156,212 with fatality
88 rates ranging from 33% to 64% 202,213-215 . LA is predominantly associated with d4T and ddI exposure 156,200,201,203,204,216-218 , but also with chronic 157,159,219,220 , neonatal 158 and perinatal 221 exposure to AZT, although in contrast to d4T, the majority (90%) of
AZT-associated LA cases were reported in the monotherapy era 202 . Host risk factors of LA include female gender, concurrent liver disease (HCV co-infection), age, severe immunodeficiency and obesity 201,202,211 .
2.1.2.4 NEUROPATHY
Distal sensory neuropathies which include HIV-1-associated sensory neuropathies
(HSN) and antiretroviral-associated neuropathy (AAN) remain common during the
HAART era, affects between 30% and 70% of HIV-1+ HAART recipients 222-225 and results in significant reductions in health-related quality of life 226 . Clinically, electrophysically and histologically, HSN and AAN are difficult to distinguish
(reviewed by Keswani et al 227 ). HSN and AAN are characterised clinically by peripheral pain and dysaesthaesia, with rare motor involvement; electrophysiologically by diminished or absent sensory action potential amplitudes with normal or slightly slowed motor conduction velocities 228 ; and histologically by a ‘dying-back’ axonopathy and axonal degeneration of myelinated and unmyleinated fibres 229 . However, consistent with a mitochondrial toxicity, it has been reported that raised serum lactate levels can discriminate AAN from HSN with 90% sensitivity and specificity 230 .
Risk factors for HSN and AAN include AIDS, age (older subjects), alcohol abuse/dependence and use of d4T and ddI 231-233 . From the large Johns Hopkins AIDS
89 Service cohort study (n=1116), ddI and d4T were associated with crude incidence rates of 6.8 and 1.4 cases per 100 person-years; concurrent use of these drugs further increased risk to 3.5-fold relative to ddI alone 162 .
2.1.2.5 LIPOATROPHY
Lipoatrophy, the pathological loss of subcutaneous fat tissue, is a component of the
‘lipodystrophy’ syndrome, which also includes metabolic complications such as
dyslipidaemia and insulin resistance and often accompanied by visceral/abdominal
fat accumulation (reviewed by Nolan & Mallal 198,234 ). Lipoatrophy is highly prevalent among HIV-1-infected individuals (38% compared to 5% for healthy controls) 235-237 , predominantly associated with NRTI therapy; NRTI therapy alone is sufficient to cause lipoatrophy 238-240 , and is an independent risk factor for its occurrence in HAART recipients 241-247 . Among NRTI recipients, prevalence of
lipoatrophy is as high as 57%248 .
Lipoatrophy is observed predominantly in d4T recipients, and to lesser extent AZT recipients, with risks of clinically apparent lipoatrophy of 40-50% among d4T recipients and 10-20% among AZT recipients 248-250 (Figure 2.4). Studies
investigating the role of ddI and 3TC in development of lipoatrophy failed to
demonstrate associations 248-250 and similarly, clinical trials of less mitochondrial
toxic NRTIs such as abacavir, tenofovir and emtricitabine show no association with
lipoatrophy 150,251,252 . Furthermore, substitution of d4T and AZT to abacavir 253-258 and even d4T to AZT or abacavir 259-261 has been demonstrated to halt progression and even reverse lipoatrophy.
90
Figure 2.4 Proportion of patients with peripheral fat depletion according to each body area, in the zidovudine and stavudine arms. Unshaded bars - zidovudine arm; shaded bars - stavudine arm. * - means significantly different from zidovudine arm (P<0.05). Taken from reference 250 (reprinted by permission from Lippincott Williams & Wilkins) [AIDS , 2002;16:2447-54].
2.1.2.6 PANCREATITIS
Pancreatitis is characterised clinically by abdominal pain, nausea and vomiting, and biochemically by elevations of lipase/ and or amylase. From a large meta-analysis study by Reisler and colleagues262 of twenty Adult AIDS Clinical Trials Group
(AACTG) studies involving 6,287 individuals, incidence rates (per 100 person years) of pancreatitis ranged from 0.47-0.79 based on clinical diagnoses, and 1.95-2.55 based on laboratory diagnoses. The clinical incidence rates, while likely underestimating the incidence rates, provide specificity regarding diagnosis, as hyperamylasemia and hyperlipasemia are commonly associated with conditions such as renal insufficiency, liver disease and cancer 262 .
Pancreatitis is predominantly associated with combined ddI and d4T therapy 263 , with little evidence of a contribution by AZT. The mean clinical incidence rate of pancreatitis from the previously mentioned meta-analysis 262 was 4.16 per 100 person
91 years (PY) for ddI/d4T regimens, compared to 0.38 and zero PY for ddI and d4T monotherapy, respectively. Regarding AZT, incidence rates ranged from zero PY for
AZT/3TC, AZT/d4T and AZT/3TC/ddC to 0.28 PY for AZT monotherapy and 1.70
PY for AZT/ddI/nevirapine.
The risk of pancreatitis associated with ddI is highlighted from studies of concomitant ddI and tenofovir (TDF) use, which increases plasma ddI concentrations by 60% 264 . Several case studies of acute pancreatitis in individuals receiving ddI/TDF therapy 265,266 and significantly higher incidence rates of pancreatitis with
ddI/TDF regimens (2.3 PY) compared to ddI without TDF (0.5 PY) and TDF
without ddI (0 PY) in a Spanish cohort have been reported 267 .
There have also been case reports of pancreatitis associated with ritonavir- and nelfinavir-containing regimens 268-270 . In the ritonavir cases, pancreatitis was likely to
be secondary to hypertriglyceridaemia, which is commonly associated with ritonavir
therapy (reviewed by Lee et al 271 and Nolan 272 ) and a known risk factor for pancreatitis (Bae et al 273 ; reviewed by Yadav & Pitchumoni 274 ). In the nelfinavir case, the pancreatitis that developed initially and in response to a rechallenge with nelfinavir occurred in the absence of hypertriglyceridaemia 268 .
Additional risk factors reported for pancreatitis include lamivudine, older age,
female sex, symptomatic HIV-1 infection, substance and alcohol abuse, and liver and
cardiovascular diseases 160,275-277 .
92 2.1.3 MECHANISMS OF THYMIDINE -ANALOGUE INDUCED
MITOCHONDRIAL DYSFUNCTION
The adverse clinical outcomes associated with thymidine-analogue therapy described in section 2.1.2, or indeed, associated with NRTI therapy in general, are believed to be due to mitochondrial dysfunction 246,278-282 . The mechanisms by which the thymidine-analogues AZT and d4T induce mitochondrial dysfunction are primarily mtDNA-mediated, although the haematological toxicities associated with AZT, appear to involve mtDNA-independent mechanisms.
2.1.3.1 MITOCHONDRIAL DNA DEPLETION
Simpson and colleagues 283 first proposed impaired mtDNA replication, leading to insufficient production of the 13 mtDNA-encoded protein subunits of the respiratory chain and resulting in insufficient energy production and cellular toxicity, as a mechanism of AZT toxicity and mitochondrial dysfunction.
The mechanism by which NRTIs induce mtDNA depletion is unclear. In vitro , most
NRTIs directly inhibit the mitochondrial-specific DNA polymerase (pol-γ) and mtDNA synthesis at pharmacological concentrations, with a consistent order of potency ddC >> ddI > d4T > AZT > ABC = TDF 150,252,284,285 . Importantly 3TC, abacavir and tenofovir, which are not associated with mitochondrial toxicity, do not significantly inhibit mtDNA synthesis at pharmacological doses. In support of this, reduced mtDNA copy number per cell has been observed in various cell types following AZT or d4T exposure in vitro , including HepG2 cells 280 and rat skeletal
93 myocytes 286 and in vivo in rat cardio(myocytes) and adipose tissue 164,287-289 , heart,
skeletal muscle, cerebellum, and cerebrum of transplacentally exposed Erythrocebus
patas monkeys 290 , muscle of perinatally-exposed infants 291 , and adipose tissue of lipoatrophic AZT-recipients 292-294 (Figure 2.5).
Clinically, mtDNA depletion has been observed in cases of NRTI-associated
myopathy 165,295 , neuropathy 296 , lactic acidosis, hyperlactataemia 157,297,298 and
lipoatrophy 299-301 . Moreover, the degree of NRTI-induced mtDNA depletion is
significantly correlated to the degree of lipoatrophy in longitudinal analyses, with
d4T therapy being associated with both significantly greater mtDNA depletion and
significantly greater relative risk of lipoatrophy compared to AZT therapy 302 . In addition, utilising multivariate models, the univariate association of duration of d4T and AZT therapy with lipoatrophy was lost after adjustment for the effect of mtDNA concentrations, suggesting that the association between choice of NRTI (d4T>AZT) and lipoatrophy is mediated through mtDNA concentrations 302 .
94
A
B
Figure 2.5 The effect of AZT on mtDNA/ nDNA ratio in vitro and in vivo . (A) AZT exerts a concentration-dependent effect on the mtDNA/nDNA ratio (expressed as a percent of untreated controls) of CD34+ haematopoietic progenitor cells cultured for 12 days. Taken from reference 303 (reprinted by permission from Hodder Arnold Ltd) [ Hum Exp Toxicol , 2004;23:173-85]. (B) AZT recipients have significantly reduced adipocyte mtDNA/nDNA ratios compared to antiretroviral-naïve individuals. Taken from reference 294 (reprinted by permission from Lippincott Williams & Wilkins) [AIDS, 2003;17:1329-38].
95 2.1.3.2 NON -MITOCHONDRIAL DNA DEPLETION MECHANISMS OF AZT
INDUCED MITOCHONDRIAL TOXICITY
While depletion of mtDNA following AZT exposure has been reported in neuronal and myocyte cell lines, only one study has detected mtDNA depletion in erythroid cells, either in vitro or in vivo 303 . Thus, AZT-associated haematological toxicities are unlikely to be mediated by mtDNA depletion. Indeed, in vitro cytotoxicity is closely related to the concentration of AZT monophosphate (AZT-MP) rather than the AZT triphosphate (AZT-TP) 304 . In the absence of mtDNA depletion, AZT exerts almost immediate effects on primary human lymphocyte proliferation 305 and a marked yet reversible reduction in mitochondrial membrane potential of rat myotubes 306 . AZT induces an increased production of ROS, which in rat heart mitochondria, resulted in inhibition of the inorganic phosphate (Pi) translocator that affects the distribution of flux control among the enzymes of oxidative phosphorylation 307 . In isolated rat liver mitochondria, AZT inhibited the ADP/ATP antiport 308 and adenylate kinase 309 which may contribute to the ATP deficiency observed in AZT exposed cells and mitochondria 282 . That AZT did not inhibit the Pi translocator in rat liver
mitochondria highlights the tissue specificity in AZT sensitivity 307 . Thus, AZT exerts an effect on oxidative phosphorylation, which may be mediated by mtDNA depletion and/or inhibition of key oxidative phosphorylation enzymes.
Increased production of reactive oxygen species (ROS) is a common observation of cells cultured in the presence of AZT 310,311 and a higher urinary excretion of 8-oxo-
7’8-dihydro-2’deoxyguanosine, a common marker of oxidatively damaged DNA, has
been reported in AZT-treated HIV+ patients compared with HIV+, AZT-naïve
96 controls that could be prevented with co-administration with antioxidants, vitamin C and E 312 . In addition, significant increases of 8-oxo-2’-deoxyguanisine in nuclear
DNA has been reported in mice following transplacental exposure to AZT 313 .
2.1.4 NON -MITOCHONDRIAL DYSFUNCTION MECHANISMS OF AZT
TOXICITY
While it is clear from the overwhelming evidence that mitochondrial dysfunction is central to NRTI toxicity in general, non-mitochondrial mechanisms contribute, at least in part, to the haematological toxicities and carcinogenesis associated with AZT therapy. These shall be discussed below.
2.1.4.1 GENOTOXICITY
Consistent with the known mechanism of AZT as a DNA chain terminator, AZT is known to be genotoxic in mammalian cells 314 . The genotoxicity of AZT includes gene mutations, sister chromatid exchanges, micronucleus formation, chromosomal aberrations, and cell transformation 315 . Incorporation of AZT into DNA of human bone marrow 316,317 , T-lymphoblastoid cells 318 and multiple organs of mice and primates, including liver, uterus, spleen and kidney 319,320 has been reported. In humans, incorporation of AZT into DNA was detected in 70% of peripheral leukocyte and cord blood DNA samples from HIV-1+ pregnant women and their in utero exposed infants, respectively, at concentrations up to 183 and 344 molecules of
AZT/10 6 nucleotides, respectively, with exposure times ranging from 10 days to 9 months 321 . Even with only 4 hours of in utero exposure to equivalent pharmacological doses of AZT, DNA incorporation of AZT at rates between 29 and
97 1944 molecules of AZT/10 6 nucleotides was detected in liver, lung, heart, skeletal muscle, brain and testis of foetal Rhesus macaca mulatta monkeys which exhibit similar AZT pharmacokinetics to pregnant humans 322 .
DNA incorporation of AZT has been proposed by Sommadossi and colleagues 317 as
a mechanism of NRTI-associated haemotoxicity. As mentioned previously,
haematological toxicities are predominantly associated with AZT therapy, and while
macrocytosis does occur in d4T recipients, its severity is significantly lower
compared to AZT therapy, consistent with in vitro data that indicate d4T is 20-100-
fold less toxic than d4T in a human CFU-GM clonogenic assay 323 . Sommadossi et al 317 proposed that this is due to the observed 10-50 fold lower steady state levels of d4T incorporation into human bone marrow DNA relative to AZT. This was not due to differential incorporation rates per se , as d4T-TP and AZT-TP have similar affinities for human nuclear DNA polymerases, but to enzymatic excision of incorporated d4T, which was not detected with AZT. The failure of AZT to be excised from DNA was attributed to the high intracellular concentrations of AZT-
MP, which is known to inhibit the 3’ to 5’ exonuclease activity of DNA polymerases 324 and while it is unclear if d4T-MP also inhibits 3’ to 5’ exonuclease activity of DNA polymerases, intracellular concentrations of d4T-MP are low and therefore unlikely to have an influence on the excision process 316 . Such a mechanism
of AZT toxicity is consistent with data that suggest AZT toxicity is associated with
AZT-MP concentrations, rather than AZT-TP concentrations.
98 2.1.4.2 INHIBITION OF HEME SYNTHESIS
Current evidence indicates that AZT directly inhibits erythroid differentiation via multifactorial mechanisms. Spiga and colleagues 325 have provided evidence that
AZT specifically inhibits the binding level of the erythroid-specific transcription factor GATA-1 at concentrations as low as 0.1 µM. In primary erythroid blast forming unit (BFU-E) cells cultured in the presence of pharmacologically relevant concentrations of AZT (1 µM) for 14 days, binding levels of GATA-1 were reduced in a dose-dependent manner and resulted in a 5-fold reduction in steady state levels of β-globin mRNA (Figure 2.6) (due to a decrease in gene transcription rather than mRNA stability) and 47% reduction in haemoglobin concentrations compared to controls.
Figure 2.6 The effect of AZT on cell growth and haemoglobin production. Human erythroid progenitor cells were cultured in the presence or absence of 1 mM AZT. Cell number and haemoglobin production were determined after 14 days in culture. Each value is the mean ± S.D. of the value obtained from at least three different experiments. Taken from reference 325 (reprinted by permission from Elsevier Science) [ Antiviral Res , 1999;44:167-77).
99 The inhibitory effect of AZT on β-globin transcription was specific and was not due
to a temporal inhibition of differentiation, as AZT has no detectable effect on mRNA
expression of other genes involved in heme biosynthesis including erythroid-specific
5-aminolevulinic (ALAS-E) and porphobilinogen (PBGD-E), which are normally
upregulated during the differentiation process earlier and later than β-globin, respectively 326 . However, inhibition of ALAS activity (71%) has been reported in rat
bone marrow cells exposed to pharmacologically relevant concentrations of AZT 327 .
2.1.4.3 NUCLEOTIDE POOL IMBALANCE
Manipulation of the intracellular nucleotide pool composition has also been shown to
reduce the toxicity exerted by NRTIs. The effect of administration of NRTIs on
intracellular nucleotide pools is uncertain. Twenty four hour treatment of
osteosarcoma cells with 0.1 µM AZT has been demonstrated to induce a 60%
depletion of the dTTP pool, although the relevance of this is unknown 328 . That the administration of 100 µM of β-D-uridine (an endogenous natural nucleoside), but not
β-L-uridine or α-D-uridine, protected against the toxic effects of AZT, 3’-fluoro-3’- deoxuthymidine (FLT) and the toxic AZT metabolite 3’-amino-3’deoxythymidine
(AMT) in granulocyte-macrophage colony-forming units (CFU-GM), indicates that
β-D-uridine had to be metabolised to nucleotides to produce a pharmacological effect 329 . The observation that 50 µM of uridine achieved complete reversal of AZT
toxicity in vitro , greater than that achieved with cytidine, suggested that the
conversion of cytidine to uridine, mediated by cytidine deaminase, is required for the
reversal of AZT toxicity by cytidine; 100 µM of thymidine can also protect against
AZT toxicity in vitro . As the deamination of cytidine to uridine, followed by
100 methylation of uridine, is the dominant pathway by which thymidine is synthesised intracellularly, the above evidence suggests that AZT toxicity is a consequence of a dTTP deficiency. Coadministration of non-toxic concentrations of thymidine with
AZT however, reduces the anti-HIV activity of AZT by 50%, probably due to a decrease in the ddNTP: dNTP ratio. Uridine however, could be administered at concentrations several-fold higher than thymidine without interfering in the anti-HIV activity of AZT 330 . Therefore, AZT may induce a dTTP deficiency that is associated with AZT toxicity.
2.1.5 PHOSPHORYLATION OF AZT & D4T
Each of the mechanisms of NRTI toxicity described above is dependent on NRTI phosphorylation as NRTIs have no intrinsic activity and require sequential phosphorylation to their respective 5’-mono-, 5’-di- and 5’-triphosphate anabolites by cellular kinases in order to exert both antiviral activity and toxicity 136,331 . Thus, the efficiency which the cellular kinases responsible for nucleotide phosphorylation phosphorylate NRTIs is likely to be central to NRTI toxicity. As depicted in Figure
2.7, upon entering the cell by passive diffusion 332,333 , AZT and d4T phosphorylation is achieved by salvage pathway enzymes thymidine kinase (TK) 1 and/or 2, deoxythymidylate kinase (dTMPK) and the base-unspecific nucleoside diphosphate kinase (NDK), respectively.
101
Figure 2.7 TMPK is at the junction of the de novo and salvage pathways of dTTP synthesis. AZT and d4T are first phosphorylated by the salvage pathway enzyme thymidine kinase 1 and 2, and subsequently by dTMPK and nucleoside diphosphate kinase to yield the antiviral triphosphate forms. Taken from reference 335 (reprinted by permission from Bentham Science Publishers Ltd) [ Mini Rev Med Chem , 2004;4:351-9].
2.1.5.1 THYMIDINE KINASE 2
As mentioned above, the initial phosphorylation of AZT and d4T is catalysed by thymidine kinase 1, the cytosolic, S-phase specific thymidine kinase, and thymidine kinase 2, the constitutively expressed, mitochondrial isozyme 334-337 . Thus, in proliferating cells, AZT and d4T are predominantly phosphorylated by TK1, while in resting or mitotically quiescent cells, AZT and d4T are predominantly phosphorylated by TK2.
102 This initial phosphorylation of the free-drug is the rate-limiting step in d4T anabolism. As shown in Figure 2.8, the free-drug form of d4T is the major d4T form in vivo , accounting for approximately 90% of all d4T metabolites, with approximately equal concentrations of the -DP and -TP metabolites 338-340 . This is attributed to the very low affinities of TK1 and TK2 for d4T, which compared to thymidine, phosphorylate d4T at efficiencies of only 7% and 1%, respectively 336,339-
342 . Therefore, TK2 is likely to be the rate limiting enzyme for d4T anabolism in mitotically quiescent cells, cells which are typically affected by d4T toxicities.
Figure 2.8 Comparison of phosphorylation of 2 ,uM D4T with 2 F,M AZT in CEM cells. Cells were incubated for 24 h with labelled drug and then processed to quantitate intracellular metabolites. Taken from reference 342 (reprinted by permission from American Society for Microbiology) [Antimicrob Agents Chemother , 1989;33:844-9].
The role of TK2 genetic variation to d4T toxicities has not been investigated.
Numerous mutations in the human TK2 gene have been associated with myopathic forms of mtDNA depletion syndrome, highlighting the role of TK2 in maintenance of mtDNA copy number and the tissue specificity of both mtDNA depletion
103 syndromes and TK2 deficiencies. Such mutations include T77M, R161K, T64M,
R183W, H121N and I212N. The T77M, R161K and I212N mutations have been shown to reduce TK2 activity 80-99% in vitro compared to wild type TK2 343-345 resulting in reductions in mtDNA copy numbers in affected muscle to approximately
92% compared to unaffected organs 344 . While the described TK2 mutations are
typically lethal and thus unlikely to play a role in susceptibility to d4T toxicities, the
potential role of novel, non-lethal TK2 polymorphisms remains to be determined.
2.1.5.2 DEOXYTHYMIDYLATE KINASE
In contrast to d4T metabolism, the phosphorylation of AZT-MP to AZT-DP
catalysed by deoxythymidylate kinase (dTMPK) is the rate-limiting step for AZT
activation. AZT-MP is the major AZT metabolite in vivo and represents
approximately 95% of all phosphorylated forms 337,346,347 . Concentrations of AZT-DP
and AZT-TP are approximately equal, accounting for approximately 0.5-3% of total
AZT intracellular metabolites in stimulated and resting PBMCs at clinically relevant
AZT concentrations 348,349 . AZT-MP exerts negative feedback on dTMPK by
reducing the catalytic rate constant (k) by approximately 200-fold and by inhibiting
the substrate binding of adenosine triphosphate, the phosphate donor to the
enzyme 350 . Such inhibition of dTMPK, together with the AZT-mediated competitive
inhibition of TK1 and TK2, reduces phosphorylation of dT 351 , further accentuating
the AZT-TP:dT-TP ratio and consequent antiviral efficacy. Although constitutively
expressed, dTMPK activity is negatively associated with cell cycle progression.
dTMPK activity in dividing 3T3-L1 adipocytes was 1049 pmol/(mg/min) and
104 decreased in resting 3T3_L1 and adipocytes 4.8- and 2.7-fold, respectively although this has not been confirmed in other studies 152 .
AZT-MP concentrations are positively associated with AZT cytotoxicity.
Cytotoxicity, determined by the MTT assay, was closely related to intracellular concentrations of the AZT-MP but not with the active metabolite AZT-TP in various cell lines (Figure 2.9). Furthermore, AZT-MP concentrations are significantly higher in patients with CD4 + T cell counts < 100 x 10 6 cells/L 349,351 and may explain the well known occurrence of increased AZT toxicity in patients with more advanced disease 349 .
The inefficiency of phosphorylation of thymidine-analogues by dTMPK is due to a combination of the size and hydrogen bonding abilities of the 3’ substituent and its effect on the sugar ring conformation (for review see Lavie & Konrad, 2004 352 ). In the case of AZT, the substitution of the 3’-hydroxyl for the bulky nonreactive azido group results in a 60-fold reduction in the rate of phosphorylation compared to that of dT-MP phosphorylation 353 . From studies of yeast 354 and human 355 dTMPK it has been demonstrated that the azido group reduces phosphorylation because it interferes with the conformational change in the phosphate binding loop (P-loop) required for binding the phosphoryl donor (ATP) to the phosphoryl acceptor (dT-MP) via the 3’- hydroxyl group, causing the P-loop to remain in the ‘open’ conformation rather than the ‘closed’ conformation observed with dT-MP.
105
Figure 2.9 The relationship between MTT cytotoxicity (bars) and AZT-MP and AZT-TP (trend lines) in five cell lines. Histogram shows percentage decrease in MTT absorbance following a 72-h incubation with AZT (100 mM). Intracellular levels of AZT-MP and AZT-TP following a 24-h incubation with AZT (0.1 mM; 1.2 mCi). All data from n=4 experiments (mean±SD). Taken from reference 305 (reprinted by permission from Elsevier Science) [ Toxicol Appl Pharmacol , 2001;177:54-8]. d4T-MP is phosphorylated to d4T-DP by dTMPK relatively efficiently, with a rate one fourth of that observed with dT-MP 356 . Compared to AZT-MP, d4T-MP is a
better substrate for dTMPK because it allows dTMPK to form a ‘partially-closed’
conformation, which is in part attributed to the conformation of the ribose moiety.
With all thymidine analogues tested, the ribose moieties are in a 3’-endo
conformation, which allows their phosphate group to makes two interactions with
Arg97 (to N ε and NH2) via two of its phosphate oxygens. In contrast, the ribose
106 atoms of d4T-MP are in plane due to the double bond between atoms C2’ and C3’ which causes the phosphate group to shift away from Arg97, breaking the interaction with the NH2 atom and moves within 3.1 Å to Arg45. The Arg97 NH2 atom that previously made an interaction with the phosphate group forms an H-bond with
Asp15 of the P-loop which stabilises the ‘partially-closed’ conformation 356 .
Despite the role of dTMPK in AZT-MP phosphorylation and the association of
AZT-MP concentrations with AZT toxicities, no data exists regarding the role of dTMPK genetic variation in AZT metabolism and associated toxicities.
2.1.5.3 NUCLEOSIDE DIPHOSPHATE KINASE
The conversion of di-phosphates to tri-phosphates catalysed by NDK is not considered rate-limiting because NDK is expressed at high levels in cells at exhibits broad substrate specificity. All NDK isoforms (eight isoforms in total, the cytosolic isoforms A and B contribute approximately 80% of the total cellular activity and the mitochondrial form contributes the remaining 20%) are base-unspecific but exhibit steady state efficiencies in the order of dG-TP > dA-TP = dT-TP >> dC-TP. d4T and
AZT diphosphates however, are catalysed at efficiencies 5,000-10,000 fold lower than the natural substrates, due to a 500-1000-fold reduction in phosphotransfer rate and 10-fold reduction in affinity 357. The inefficiency of AZT and d4T catalysis is due to the absence of the 3’-OH group, which normally forms a hydrogen bond network with Lys12 and Asn115 of the NDK amino acid side chains involved in catalysis
(Figure 2.10).
107
AB
Figure 2.10 The reduced phosphorylation of nucleoside analogues is due to the absence of the 3’-OH group which reduces the catalytic efficiency rather than the binding affinity. (A) View of human NDK isoform B active site with bound dTDP (pruple). Some amino acid side chains are shown in yellow. The 3’-OH of the ribose H-bonds with Lys12 and Asn115 side chains. (B) Correlation of catalytic efficiencies of NDK for several nucleotides triphosphate and analogues and their binding efficiencies. Substrates were found to belong to two classes according to the presence of a 3’-OH group which corresponds to a difference in catalytic efficiencies of three orders of magnitude. Taken from reference 358 (reprinted by permission from Bentham Science Publishers Ltd) [ Mini Rev Med Chem , 2004; 4:361-9].
108 2.1.6 HYPOTHESES & AIMS
2.1.6.1 HYPOTHESIS
We hypothesised that genetic variation in thymidine kinase 2 (TK2) and deoxythymidylate kinase (dTMPK) would influence the efficiency of phosphorylation of AZT and d4T, respectively, which would be reflected in mtDNA levels and haematological parameters in response to such therapy.
2.1.6.2 AIMS
1. To identify single nucleotide polymorphisms (SNPs) and characterise
haplotypes of the human TK2 and dTMPK genes.
2. To investigate whether TK2 and dTMPK SNPs and/or haplotypes influence
adipocyte mtDNA concentrations and haematological parameters in response
to AZT and d4T therapy, respectively, in HIV-1+ individuals.
109 2.2 MATERIALS & METHODS
2.2.1 IDENTIFICATION OF SNP S AND CHARACTERISATION OF HUMAN
TK2 & D TMPK HAPLOTYPES
Identification of human TK2 and dTMPK single nucleotide polymorphisms (SNPs), and characterisation of dTMPK genetic haplotypes, was performed using a pooled
DNA sample and DNA from 8 parent-pairs, respectively. The pooled DNA sample comprised of DNA from 187 northern European individuals and has been used previously by others to estimate allele frequencies118 . Estimation of allele frequency
from the pooled DNA sample was achieved by chromatogram relative peak height.
Oligonucleotide primers were designed to allow PCR amplification and sequencing
of the TK2 and dTMPK exonic and flanking intronic regions, the putative 5’
promoter region (up to approximately 2.5kb 5’ of transcription start site (TSS)) and
0.5kb 3’ of the 3’ untranslated region (UTR). Optimisation of the PCR amplification
of each dTMPK and TK2 amplicon was achieved by adjusting MgCl 2 concentrations and annealing temperature. The PCR products were then purified using Exosap ® according to the manufacturers protocol and sequenced using M13 forward and reverse primers (and overlapping primers for the dTMPK 5’ putative promoter regions) and the Big Dye Terminator kit, version 3.3 (PE, Applied Biosystems,
USA). Sequences were analysed using Assign SBT software v. 3.2.7 (Conexio
Genomics, Australia).
110 2.2.1.1 AMPLIFICATION OF DTMPK
The human dTMPK gene, located on chromosome 2q37.3, is comprised of 5 exons spanning approximately 11 kb. For amplification of the dTMPK exonic and flanking intronic sequence, 2 µL of DNA was amplified in a volume of 50 µL containing 10X
PlatinumTaq PCR buffer, 2mM MgCl 2 (4mM MgCl 2 for exon 1), 0.8mM dNTP mix,
100nM of each forward and reverse primer and 1U of PlatinumTaq under the following conditions; 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 70 °C for 90 s and a final incubation at 72 °C for 10 min. For amplification of the putative dTMPK 5’ promoter region, 2 µL of DNA was amplified in a volume of 50 µL containing 10X PCRx buffer, 0.8mM dNTP mix,
1.5mM MgSO 4, 100nM of each forward and reverse primer (Table 2.1), 1X PCRx
Enhancer solution and 0.5U of PlatinumTaq under the following conditions; 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s and 68 °C for 3 mins.
111 Table 2.1 Oligonucleotide characteristics used for the amplification and sequencing of the human dTMPK gene.
Primer 5’ Binding Region Orientation Sequence (5’-3’) * Name Position 103,755F † Forward 25,401 aaaccgcacagttcacagc 106,366R † Reverse 22,790 ggccacaagacagaaactcc 104,504R ‡ Reverse 24,653 catctcgctcgttcgtctc 5’ Prom 104,485F ‡ Forward 24,672 cgagacgaacgagcgagat 105,213R ‡ Reverse 23,944 tagataccggggacacttcg 105,105F ‡ Forward 24,052 tctttccatgcctccaactc 105,920R ‡ Reverse 23,237 agggtgcagtgagctgagat Exon 1 103,619F Forward 25,537 gcctatgttcccggctacc 104205R Reverse 24,951 ccaggacacggctccagt Exon 2 102865F Forward 26,290 aacatcaggcccagcactac 102865R Reverse 25,920 gcagaagatccaagcctgtc Exon 3 97350R Forward 31,806 aatctgaggcatgtcactcg 97350R Reverse 31,542 gcacagcctgttgggataat Exon 4 95543F Forward 33,613 cacatctgggaggaccagac 95543R Reverse 33,199 tcaccaaatgtcccctcatt 92891F Forward 36,264 gatggaggttcccattcaaa Exon 5 92891R Reverse 35,588 tacaagcagtcctccctcgt
* - all oligonucleotide sequences (except for 103,755F and 106,366R) contained M13F (5’- tgtaaaacgacggccagt-3’) and M13R (5’-caggaaacagctatgacc-3’) tags at the 5’ end of the oligonucleotide for sequencing practicality; 5’ binding position – nucleotide position of the 5’ end of the oligonucleotide relative to NCBI accession number AC133528; † - oligonucleotides used for both PCR amplification and sequencing of the putative 5’ promoter amplicon; ‡ - oligonucleotides used for sequencing only of the 5’ promoter amplicon.
2.2.1.2 AMPLIFICATION OF TK2
The human TK2 gene, located on chromosome 16q22-q23.1, is comprised of 9 exons spanning approximately 39 kb. For amplification all TK2 exons and their flanking intronic sequence and the putative 5’ promoter, 2 µL of DNA was amplified in a volume of 50 µL containing 10X PlatinumTaq PCR buffer, 3mM MgCl 2 (1.5mM
MgSO 4 for putative 5’ promoter) 0.8mM dNTP mix, 100nM of each forward and
reverse primer (Table 2.2) and 1U of PlatinumTaq under the following conditions;
112 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s (65 ° for 60 s for putative 5’ promoter) and 70 °C for 90 s and a final incubation at 72 °C for 10 min.
Table 2.2 Oligonucleotide characteristics used for the amplification and sequencing of the human TK2 gene.
Primer 5’ Binding Region Orientation Sequence (5’-3’)* Name Position 8680F Forward 8,680 caggtgatctgccttccttg 5’ Prom 9611R Reverse 9,611 ctctccatcccagaaccaaa 9429F Forward 9,429 ctatgcgcccacctactcc 10016R Reverse 10,016 aggcagaacagcctccact Exon 1 9836F Forward 9,836 actgttacacccgggttctg 10484R Reverse 10,484 tctccctggagatcctcttgt 17235F Forward 17,235 cgaggagggattttggttta Exon 2 17576R Reverse 17,576 tacacctgtggcttgcactt 22228F Forward 22,228 tctcctcactttcccctcaa Exon 3 22495R Reverse 22,495 ccctggtcttctccaactca 27809F Forward 27,809 tcagtgccttgtgagaccag Exon 4 28015R Reverse 28,015 aagtttcccttcctggcaat 30122F Forward 30,122 ggtcctcctgcttcactctct Exon 5 30480R Reverse 30,480 tttgttacaccgcattgcta 41328F Forward 41,328 gatcacactgtgccctggta Exon 6 41694R Reverse 41,694 cctcagggagaaagctgaact 42021F Forward 42,021 gagctgtgcagaccttcctc Exon 7 42362R Reverse 42,362 ggccaaacaggaagtcagag 45369F Forward 45,369 ctgtccacttgagccaggtt Exon 8 45778R Reverse 45,778 agactcagcccagaaacagg 47139F Forward 47,139 atctgcctgcaaccttctgt 47674R Reverse 47,674 cacaacaggtgctctcccta 47512F Forward 47,512 tctttgtcatgcgtctctgg Exon 9 48159R Reverse 48,159 aggagactgagcacccttca 47995F Forward 47,995 cccaaggacagtcagcattt 48687R Reverse 48,687 tggcctgtttctccagagtt
* - all oligonucleotide sequences contained M13F (5’-tgtaaaacgacggccagt-3’) and M13R (5’- caggaaacagctatgacc-3’) tags at the 5’ end of the oligonucleotide for sequencing practicality; 5’ binding position – nucleotide position of the 5’ end of the oligonucleotide relative to NCBI accession number AC010289.
113 2.2.2 THE INFLUENCE OF D TMPK GENETIC VARIATION ON
HAEMATOLOGICAL RESPONSE TO AZT THERAPY
The influence of dTMPK genetic variation on haematological parameters (MCV, haemoglobin and neutrophil count and percent) in response to AZT was assessed in one hundred and sixteen patients receiving AZT as part of their initial HAART regimen and forty eight d4T-experienced, AZT recipients. Forty one patients receiving d4T as part of their initial HAART regimen and eighty two AZT- experienced, d4T recipients were used as controls. All patients were male Caucasian.
Analysis of patients’ MCV and haemoglobin profiles indicated that equilibration of such values in response to AZT therapy occurred within six months of initiation of therapy (Figures 2.11 & 2.12). Consequently, mean values for each parameter in response to therapy were calculated on serial values obtained at least six months after initiation of AZT/d4T until discontinuation of the corresponding drug.
2.2.3 THE INFLUENCE OF D TMPK GENETIC VARIATION ON AZT-
INDUCED MT DNA DEPLETION
The influence of dTMPK genetic variation on AZT-induced mtDNA depletion in the context of lipoatrophy was examined in a subset of the patients for whom we had previously obtained subcutaneous adipose tissue biopsies. This subset comprised of
29 AZT recipients and 19 d4T recipients that had been receiving the corresponding thymidine-analogue therapy for 42.7 ± 34.0 and 36.2 ± 22.4 months, respectively
(P=0.468). The number of copies of mtDNA per cell was calculated from isolated adipocytes by real-time PCR quantification of the mitochondrial-encoded 16S gene and the nuclear-encoded HGH gene as previously described 294 .
114
A 140
130
120
110 100
MCV (fl) MCV 90
80
70
60 0 200 400 600 800 1000 1200 days post initiation of AZT
B 140
120
100
80
MCV (fl) (fl) MCV 60
40
20
0 0 200 400 600 800 1000 1200
days post initiation of AZT
Figure 2.11. Longitudinal mean corpuscular volume (MCV) profiles of AZT-recipients. (A) MCV profiles of individuals receiving AZT as part of initial HAART regimen. (B) MCV profiles of AZT recipients with had received previous d4T treatment. Individual on-treatment MCV values were calculated from serial values obtained at least 176 days (approximately 6 months) after initiation of AZT.
115
A 200
180 160
140 120
100
(fl) Hb 80 60
40 20
0
0 200 400 600 800 1000 1200
days post initiation of AZT
B 200
180 160
140 120
100
Hb (g/L) (g/L) Hb 80 60
40 20
0 0 200 400 600 800 1000 1200
days post initiation of AZT
Figure 2.12. Longitudinal haemoglobin concentration profiles of individuals initiating AZT therapy. (A) Haemoglobin profiles of individuals receiving AZT as part of initial HAART regimen. (B) Haemoglobin profiles of AZT recipients with had received previous d4T treatment. Individual on- treatment haemoglobin values were calculated from serial values obtained at least 176 days (approximately 6 months) after initiation of AZT; Hb – haemoglobin.
116 2.2.4 STATISTICAL ANALYSIS
Statistical analyses were carried out using SPSS software (version 12.0.1, SPSS Inc.
USA). All values are expressed as mean ± standard deviation unless otherwise stated. Comparisons of means were performed using T-test or ANOVA and adjustment for multiple comparisons where appropriate.
117 2.3 RESULTS
2.3.1 IDENTIFICATION OF TK2 AND D TMPK SNP S
As mentioned in section 1.2.7, use of a pooled DNA sample allowed identification of
APOBEC3G SNPs with allele frequencies greater than approximately 10-15%.
Utilising this approach, we identified nine novel and three previously reported dTMPK SNPs (Figure 2.13). The three previously reported dTMPK SNPs identified from the pooled DNA sample include a synonymous (33,309, rs4675906), an intronic (33,508, rs4076639) and a 3’UTR (35,887, rs3209844) SNP. The nine novel dTMPK SNPs identified include four intronic (25,421, 25,424, 25,946 and 33,567), two putative 5’ promoter (24,537 and 24,880) and three 3’UTR SNPs (35,830;
35,897; 36,187).
exon 1 exon 2 exon 3 exon 4 exon 5
5’ 3’ 11.018 kb
Figure 2.13 Schematic representation of the human dTMPK gene and location of identified SNPs. Vertical line – position of observed SNPs; turquoise shading – exons; grey shading – untranslated regions; blue shading – putative 5’ promoter regions.
Regarding TK2, we did not observe any SNPs in the regions examined from the pooled DNA sample, suggesting that the putative 5’ promoter, exonic and flanking intronic regions of the TK2 gene lacks SNPs with allele frequencies greater than approximately 10%. In order to identify TK2 SNPs with allele frequencies less than approximately 10%, we sequenced the aforementioned genetic regions in the 8
118 parent-pairs used for haplotype characterisation. From this, we identified 2 intronic
SNPs, both of which were previously unreported, located at nucleotide positions
47,207 and 47,254 relative to NCBI accession number AC010289 (Figure 2.14).
Each SNP was only observed in one individual, and only in the heterozygous state, giving allele frequencies of approximately 3% and as such, TK2 genetic variation was not investigated any further.
exon 7 exon 9 exon 1 exon 2 exon 3 exon 4 exon 5 exon 6 exon 8
5’ 3’
39.056 kb
Figure 2.14 Schematic representation of the human TK2 gene and location of identified SNPs. Vertical line – position of observed SNPs; turquoise shading – exons; grey shading – untranslated regions; blue shading – putative 5’ promoter regions.
The previously reported non-synonymous (25,263, rs11555213; 33,383 rs1803267), synonymous (33,345, rs1803266) and 3’UTR (35,878, rs1803265; 35,899 rs5860;
35,961, rs1131266) dTMPK SNPs were not identified in either the pooled DNA sample or any of the 286 individual DNA samples. Of the twelve SNPs identified in the pooled DNA sample, the mean difference between the estimated allele frequencies and that determined from the 286 individual DNA samples was 5.53 ±
3.46% (range 1.31-11.85%). Thus, allele frequency estimated by relative chromatograph peak height generated from sequencing a pooled DNA sample reflects the ‘true’ allele frequency determined by individual genotype frequency. The frequency of the dTMPK 25,421 G allele determined from the 286 HIV+ individuals was 13%, which reflects the frequency estimated from the pooled DNA sample
119 (10%) and estimates the lower detection limit of allele detection by sequencing pooled DNA is below 13%.
2.3.2 CHARACTERISATION OF D TMPK HAPLOTYPES
Of the eight, parent-pairs, five provided sufficient genetic variation to characterise dTMPK haplotypes, the pedigrees of which are displayed in Figure 2.15. By genotyping the parent-pairs and their children, we identified five haplotypes, ranging in frequency from 4.5% to 37.1% (Table 2.3). The five haplotypes appear to have evolved from recombination of three distinct 3’ haplotypes, restricted by the 24,537 and 25,946 SNPs, with three distinct 5’ haplotypes, restricted by the 33,309 and
36,187 SNPs.
120
Family 1 2,3 1,1
3,1 3,1 3,1 3,1 3,1 2,1
Family 2 3,3 3,5
3,5 3,3 3,5 3,5 3,5
1,3 2,5 Family 3
1,2 3,2 1,5 1,5
Family 4 1,4 1,3
1,4 1,3 1,1 4,3
1,4 4,5 Family 5
4,4 1,4 1,5
Figure 2.15 dTMPK haplotype pedigrees. Numbers represent haplotype number in Table 2.3; squares represent males; circles represent females.
121 Table 2.3 Human deoxythymidylate kinase haplotypes identified from studies of family members.
Haplotype AC133528 rs# Ps Region Ref Seq 1 2 3 4 5 24,537 - -624 5’P C C C T T T 24,880 - -281 5’P G G G A A G 25,421 - 260 I1 T T T T T G 25,424 - 263 I1 C C C G G G 25,946 - 785 I1 T T T A A T
33,309 4675906 8,148 E 4 syn T T C C T T 33,508 4076639 8,347 I4 T T T T C C 33,567 - 8,406 I4 T T C C C C 35,830 - 10,669 3’UTR C C C C ∆C ∆C 35,887 3209844 10,726 3’UTR C C C C T T 35,897 - 10,736 3’UTR A A G G G G 36,187 - 11,026 3’UTR C C A A C C
Freq (%) - - - - 37.1 4.5 34.1 11.5 11.8 5’ Hap - - - A A A B B C 3’ Hap - - - A A B B C C
Position – nucleotide number on Genbank Accession AC133528. Haplotypes 2 and 4 appear to have evolved from recombination of haplotypes 1 and 3 and 3 and 5, respectively; Ps – nucleotide position relative to transcription start site; 5’ P – putative 5’ promoter; I1 – intron 1; E4 syn – exon 4, synonymous; I4 – intron 4; 3’UTR – 3’ untranscribed region; Freq (%) – frequency of the haplotype determined from 286 individuals.
2.3.3 HAEMATOLOGICAL RESPONSE TO AZT AND D 4T
Haematological toxicities such as anaemia and neutropenia are commonly observed amongst AZT recipients, with few reports of such toxicities amongst d4T recipients, and while macrocytosis does develop in d4T recipients, its severity is substantially lower than that observed in AZT recipients 167,179 . Consistent with this, AZT recipients in this study (n=163) had significantly lower haemoglobin (142.9 ± 13.3 g/dL vs 151.1 ± 13.1 g/dL, P<0.001), neutrophil percent (51.6 ± 8.7 vs 54.7 ± 8.3%,
P=0.003) and higher MCV (109.4 ± 6.4 fl vs 104.7 ± 7.3 fl, P<0.001) than d4T recipients (n=123) (Table 2.4).
122 Table 2.4 Characteristics of AZT and d4T recipients.
Pre-treatment AZT (n=163) d4T (n=123) P value
Viral load 4.997± 0.690 4.792 ± 0.789 0.180 CD4 + cell count 293.9 ± 229.5 303.4 ± 166.1 0.800 Haemoglobin 137.9 ± 16.5 139.2 ± 22.3 0.763 MCV 89.0 ± 4.7 88.7 ± 5.5 0.830 Neutrophil count 2.68 ± 1.18 2.92 ± 1.36 0.422 Neutrophil percent 48.8 ± 15.9 56.3 ± 12.9 0.064 On-treatment Haemoglobin 142.9 ± 13.3 151.1 ± 13.1 <0.001 MCV 109.4 ± 6.4 104.7 ± 7.3 <0.001 Neutrophil count 3.68 ± 7.4 3.67 ± 1.23 0.991 Neutrophil percent 51.6 ± 8.7 54.7 ± 8.3 0.003
+ 6 Haemoglobin , g/L; viral load, log 10 copies/mL; CD4 cell count, 10 cells/mL; MCV, mean corpuscular volume, femtolitres; neutrophil count, 10 6 cells/mL.
Of the 163 AZT recipients, 71 (44%) had haemoglobin concentrations determined within 62 days (mean 17.1 ± 17.3 days) of initiating therapy. Using these measures as estimated pre-treatment values, 37.3% of AZT-recipients experienced a decrease in haemoglobin in response to AZT, with 23.5% experiencing a reduction in haemoglobin greater than 5 g/dL. These frequencies were higher in a subset of AZT recipients (n=29) with actual day zero pre-treatment haemoglobin values (50% experienced a reduction in haemoglobin, with 46% experiencing a reduction greater than 5 g/dL). In contrast, only 12.5% of d4T-recipients with estimated pre-treatment haemoglobin values (estimated within a similar duration of initiating therapy, n=24, mean 19.1 ± 22.5 days) experienced a reduction in haemoglobin, with 41.7% of d4T recipients experiencing an increase in haemoglobin greater than 5 g/dL.
123 2.3.4 INFLUENCE OF DTMPK GENETIC VARIATION ON
HAEMATOLOGICAL RESPONSE TO ZIDOVUDINE
2.3.4.1 HAEMOGLOBIN
Given the high degree of dTMPK polymorphism, we sought to determine the influence of dTMPK genetic variation on the response of haematological parameters examined above to AZT therapy. We observed significant associations between both the synonymous 33,309 SNP and the 3’ UTR 36,187 SNP and haemoglobin concentrations in response to AZT therapy. The 33,309 C and 36,187 A alleles are haplospecific for the 3’ haplotype B, which comprises the common haplotype 3 and the rare haplotype 2. As depicted in Figure 2.16A, AZT-recipients homozygous for the 3’ haplotype B, had significantly higher on-treatment haemoglobin concentrations than non-homozygotes (n=22, 149.0 ± 8.4 versus n=132, 142.1 ±
13.8, P=0.024). This comparison was essentially a comparison of individuals homozygous for haplotype 3 versus non-homozygotes, as 20/22 of the 3’ haplotype
B homozygotes are also homozygous for haplotype 3, and indeed, such a comparison remained significant (n=20, 149.1 ± 8.8 versus n=134, 142.2 ± 13.7, P=0.031).
AZT-recipients homozygous for the 3’ haplotype B were significantly more likely
than non-homozygotes to have mean on-treatment haemoglobin concentrations
greater than 150 g/L (7/14 versus 11/54, P=0.04) while individuals homozygous for
haplotype 3 tended to be associated with on-treatment haemoglobin concentrations
greater than 150 g/L (6/12 versus 12/56, Fishers Exact, P=0.068). Furthermore, as
depicted in Figure 2.16B, 0/22 and 0/20 AZT-recipients homozygous for 3’
haplotype B or haplotype 3, respectively, compared to 22/132 (17%) or 22/134
124 (16%) of non-homozygotes, respectively, had mean on-treatment haemoglobin concentrations below 130g/L, (Fishers Exact, P=0.045 & 0.079, respectively).
A
B
P=0.079
non-homozygous homozygous (n=134) (n=20)
Figure 2.16 AZT recipients homozygous for 3’ haplotype B have significantly higher on- treatment haemoglobin concentrations (A) and were significantly less likely to have on- treatment haemoglobin concentrations less than 130 g/L (B).
125 The association of the 3’ haplotype B or haplotype 3 with higher on-treatment haemoglobin concentrations was unique to AZT therapy. As shown in Figure 2.17, d4T-recipients homozygous for either the 3’ haplotype B or haplotype 3 (all d4T- recipients homozygous for the 3’ haplotype B were also homozygous for haplotype
3) had similar on-treatment haemoglobin concentrations as non-homozygous d4T- recipients (n=24, 147.3 ± 17.0 versus n=96, 152.2 ± 11.8, P=0.099).
Figure 2.17 No association existed between the 3’ haplotype B and on-treatment haemoglobin concentrations in d4T-recipients.
From a subset of AZT-recipients with estimated pre-treatment haemoglobin concentrations (n=68), it was revealed that individuals homozygous for 3’ haplotype
B (n=14, 145.5 ± 14.5 versus n=54, 136.2 ± 16.7, P=0.061) or haplotype 3 (n=12,
144.6 ± 15.5 versus n=56, 136.8 ± 16.6, P=0.138) tended to have higher pre-
treatment haemoglobin concentrations than non-homozygotes. 7/14 and 6/12 AZT-
recipients (50%) homozygous for the 3’ haplotype B or haplotype 3, respectively,
126 had pre-treatment haemoglobin concentrations greater than 150g/L compared to only
11/54 (20%) or 12/56 (21%) of non-homozygous AZT-recipients, respectively
(Fishers Exact, P=0.068 & P=0.04, respectively). Thus, the higher mean on- treatment haemoglobin concentrations observed in AZT-recipients homozygous for the 3’ haplotype B or haplotype 3 may be due to an effect of the haplotype on pre- treatment haemoglobin concentrations rather than haemoglobin concentrations in response to AZT therapy.
When the analysis of pre-treatment haemoglobin concentrations was extended to all individuals regardless of the treatment arm they belonged to after starting treatment
(n=91), the association between lower pre-treatment haemoglobin concentrations with the 3’ haplotype B (P=0.443) or haplotype 3 homozygosity was lost (P=0.661), supporting a role for dTMPK haplotypic variation in haemoglobin concentrations in response to AZT-therapy. However, the ∆ haemoglobin values were similar in individuals homozygous for the 3’ haplotype B (Figure 2.18A, n=13, 4.1 ± 12.4 versus n=53, 5.9 ± 16.1, P=0.705) or haplotype 3 (n=11, 5.3 ± 13.2 g/dL versus n=54, 5.6 ± 15.8 g/dL, P=0.949) compared to non-homozygotes, with 5/8 (62%) and
19/51 (37%) of 3’ haplotype B homozygotes and non-homozygotes, respectively and
8/11 (73%) and 32/53 (60%) of haplotype 3 homozygotes and non-homozygotes, respectively, experiencing an increase in haemoglobin concentrations in response to
AZT (Figure 2.18B, Fishers Exact, P=1.0 and P=0.514, respectively), clearly showing no influence of the dTMPK 3’ haplotype B or haplotype 3 had no influence on haemoglobin concentrations in response to AZT-therapy.
127
A
B
non-homozygous homozygous (n=53) (n=11)
Figure 2.18 Mean ∆∆∆ haemoglobin concentrations (A) and the proportion of individuals that experienced an increase in haemoglobin concentrations in response to AZT-therapy (B) were similar in individuals regardless of homozygosity for the 3’haplotype B or haplotype 3, respectively.
128 2.3.4.2 MEAN CORPUSCULAR VOLUME
Pre-treatment mean corpuscular volume (MCV) was significantly, albeit modestly, lower in AZT-recipients positive for the 24,537 and 25,424 C alleles than individuals homozygous for the 24,537 and 25,424 T alleles (n=34, 87.8 ± 4.4 versus n=22, 90.9
± 5.0, P=0.019), and unlike the pre-treatment haemoglobin association, this association remained significant when extended to all individuals regardless of treatment arm (n=48, 88.1 ± 4.7 versus n=31, 90.5 ± 4.9, P=0.035). The 24,537 and
25,424 C alleles are haplospecific for the 5’ haplotype A, which comprises the common haplotype 1 and rare haplotype 2. Lower pre-treatment MCV was also associated with the 24,880 G and 25,946 T alleles, which like the 24,537 and 25,424
C alleles are haplotypic of the 5’ haplotype A, but also haplotypic of the 5’ haplotype
C, in all individuals (n=57, 88.4 ± 4.7 versus n=22, 90.9 ± 4.9, P=0.039) and when restricted to AZT-recipients only (n=42, 88.2 ± 4.6 versus n=14, 91.4 ± 4.8,
P=0.034). Furthermore, regarding the two most common haplotypes, pre-treatment
MCV was significantly lower in individuals homozygous for haplotype 1 than in individuals homozygous for haplotype 3 (n=12, 86.9 ± 3.8 versus n=16, 90.1 ± 4.3,
P=0.050).
However, the 5’ haplotype A had no effect on MCV in response to AZT therapy.
While individuals that carried the 5’ haplotype B or haplotype 1 had significantly lower pre-treatment MCV than those that lacked the 5’ haplotype B or haplotype 1, on-treatment MCV (n=101, 143.8 ± 13.0 versus n=53, 141.7 ± 14.1, P=0.360) and ∆
MCV (n=34, 20.9 ± 5.2 versus n=19, 18.3 ± 5.9, P=0.102) were similar amongst individuals carrying the 5’ haplotype B or not. Similarly, on-treatment MCV
129 (P=0.105) and ∆ MCV (P=0.509) were similar amongst individuals homozygous for
the two common haplotypes, 1 and 3. Thus, the 5’ haplotype B or haplotype 1 did
not influence MCV in response to AZT-therapy, but may represent a response to
HIV-1 infection per se .
2.3.5 INFLUENCE OF DTMPK SNP S AND HAPLOTYPES ON AZT-
INDUCED MT DNA DEPLETION
The influence of dTMPK genetic variation on AZT-induced mtDNA depletion was investigated in a preliminary study in a subset of patients, comprised of twenty nine
AZT recipients and nineteen d4T recipients. Two dTMPK SNP positions were found to be significantly associated with adipocyte mtDNA values. As shown in Figure
2.19, homozygosity for either the 33,567 T or 35,897 A alleles, which are haplospecific for haplotype 1, was significantly associated with higher mtDNA values than patients carrying the 33,567 C or 35,897 G alleles (n=4, 3.198 ± 0.253 log 10 copies/cell versus n=26, 2.904 ± 0.226 log 10 copies/cell, P=0.023) despite similar duration of AZT therapy (44.7 ± 53.9 versus 42.7 ± 30.5 months, P=0.912).
The effect of haplotype 1 on mtDNA values in response to thymidine-analogue therapy was specific to AZT, consistent with the role of dTMPK as the rate-limiting enzyme for AZT, but not d4T, metabolism. D4T recipients homozygous for haplotype 1 had similar mtDNA levels to d4T recipients carrying other haplotypes
(n=3, 2.285 ± 0.062 versus n=16, 2.326 ± 0.262, P=0.796) despite them tending to have shorter duration of d4T exposure (20.8 ± 17.6 versus 39.1 ± 22.4 months,
P=0.203).
130
A
B
Figure 2.19 AZT recipients homozygous for the dTMPK haplotype 1 have significantly higher adipocyte mtDNA values than non-homozygous AZT recipients (A), despite similar duration of AZT exposure (B).
131 Figure 2.20 illustrates that the level of mtDNA observed in patients homozygous for haplotype 1 reflects the values obtained from antiretroviral-naïve HIV-1+ patients
(n= 34, 3.194 ± 0.312, P=0.982), HIV-1+ patients that were not receiving antiretroviral therapy at the time of biopsy (n=5, 3.120 ± 0.141, P=0.572) or HIV-1+ patients receiving HAART regimens not containing thymidine-analogues (n=25,
3.195 ± 0.239, P=0.979), suggesting that haplotype 1 was not associated with AZT- induced mtDNA depletion.
P=0.023
Figure 2.20 mtDNA values in AZT recipients homozygous for the dTMPK haplotype 1 reflect that observed in recipients of non-thymidine-analogue therapy, HIV-1+ individuals not receiving antiretroviral therapy or HIV-1 seronegative controls. d4T – d4T-recipients; AZT (non- hm) – AZT-recipients that are non-homozygous for haplotype 1; AZT (hm) – AZT-recipients that are homozygous for haplotype 1; non AZT/d4T – recipients of antiretroviral regimens that do not contain AZT or d4T; NT – HIV + individuals not receiving antiretroviral therapy at the time of adipose tissue biopsy; ART-naïve – HIV + individuals that have never received antiretroviral therapy; HIV-1 neg – HIV-1 seronegative individuals; mtDNA (log 10 copies/mL) – mtDNA/nDNA (log 10 copies/mL).
132 2.4 DISCUSSION
In this study, we observed substantial genetic variation in the human dTMPK gene amongst Caucasian individuals. Although we did not identify any non-synonymous polymorphisms, suggesting that the dTMPK protein is highly conserved and has not been subject significant selective pressure, we did identify two putative 5’ promoter and four 3’UTR SNPs which may influence its transcription or translation, respectively. Furthermore, from the nucleotide sequence of the five dTMPK haplotypes identified, it appears that they evolved from recombination of three distinct 3’ haplotypes, extending from upstream of the 24,537 SNP to the 25,946
SNPs, with 3 distinct 5’ haplotypes, restricted by the 33,309 SNP and extending 3’ of the 36,187 SNP. In contrast, the human TK2 gene was highly conserved, with only two intronic polymorphisms identified at frequencies of 3%.
The two most common dTMPK haplotypes were associated with higher pre- treatment haemoglobin concentrations and maintained adipocyte mtDNA concentrations in response to AZT therapy, respectively. The influence of dTMPK genetic variation on haemoglobin concentrations in this HIV-1 cohort was modest.
The 33,309 C allele and the 36,187 A allele, both of which are haplospecific of the
3’ haplotype B, shared by the common haplotype 3 and the rare haplotype 2, was significantly associated with higher haemoglobin concentrations in response to AZT therapy (P=0.024), with homozygous AZT-recipients significantly more likely to experience mean on-treatment haemoglobin concentrations greater than 150 g/L
(P=0.04) and non-homozygotes more likely to experience mean on-treatment haemoglobin concentrations less than 130 g/L (P=0.045). However, the higher pre- treatment haemoglobin concentration in 3’ haplotype B homozygotes was likely due
133 to higher pre-treatment haemoglobin concentrations (P=0.061), as homozygotes tended to be more likely than non-homozygotes to also have mean pre-treatment haemoglobin concentrations greater than 150 g/L (P=0.068), and this was reflected in the similar ∆ haemoglobin concentrations amongst homozygotes and non-
homozygotes (P=0.705).
The influence of the 3’ haplotype B on pre-treatment haemoglobin concentrations
suggests that the 3’ haplotype B may influence haemoglobin concentrations in
response to HIV-1 infection per se or physiological haemoglobin concentrations,
rather than a response to treatment. Haemoglobin concentrations are known to
correlate with disease progression 358-361 and baseline haemoglobin concentrations are
significant predictors of AIDS and death 176 . As little as 10 g/L decrease in haemoglobin concentrations has been associated with increased hazard of death by
57% in AIDS patients 174 . In this cohort, although significant correlations existed between pre-treatment viral load and pre-treatment haemoglobin concentrations (r=-
0.251, P=0.032, data not shown) and between pre-treatment viral load and pre- treatment MCV (r=-0.329, P=0.004, data not shown), which was also significantly, albeit modestly, higher in individuals homozygous for haplotype 3 (P=0.05), pre- treatment viral loads were similar amongst individuals homozygous for the 3’ haplotype B and non-homozygotes (P=0.355, data not shown). Therefore, although the clinical relevance of dTMPK haplotyping was limited in the relatively haematologically-stable WA HIV-1 cohort, in third world populations where up to
80% of individuals with HIV-1 present with anemia 360,362 and where routine
monitoring of haemoglobin concentrations prior to commencement of highly active
antiretroviral therapy is recommended to ensure that mortality and morbidity are
134 minimized and quality of life optimized 362 , dTMPK haplotyping may contribute, at least in part, to identifying individuals that are least likely to experience haematological complications of AZT therapy. In this respect, it would be informative to determine the role of dTMPK genetic variation on such complications in large African clinical trials, such as the DART (Development of AntiRetroviral
Therapy in Africa) study which aims to investigate whether intermittent rather than continuous AZT-containing regimens alleviate toxicity while maintaining virological control from 3,300 participants over 5 years (http://www.ctu.mrc.a c.uk/dart/summary.asp).
The results of the pilot study (n=48) regarding the role of dTMPK in AZT-induced mtDNA depletion are encouraging, but due to the small number of samples analysed, interpretations of the data are limited. Homozygosity for haplotype 1, the most common dTMPK haplotype observed in this Caucasian cohort, was significantly associated with higher adipocyte mtDNA concentrations than non-homozygotes
(P=0.023). The mtDNA concentrations observed in individuals homozygous for haplotype 1 reflected those observed in antiretroviral-therapy naïve and HIV-1 seronegative individuals and thus appear to protect against AZT-induced mtDNA depletion. Whether the haplotype protects against toxicities associated with AZT- induced mtDNA depletion remain to be determined. The lack of an effect of haplotype 1 in d4T-induced mtDNA depletion (P=0.796) was expected and consistent with data that indicate that dTMPK is able to phosphorylate d4T-MP at greater efficiency than AZT-MP and consequently, and in contrast to AZT, is not rate-limiting for d4T anabolism 356 .
135 The mechanism by which the dTMPK haplotypes influence mtDNA and haemoglobin concentrations is unclear. It would be of great interest to determine in future studies whether the SNPs located in the putative 5’ promoter and 3’UTR regions alter the expression and translation of dTMPK mRNA and whether they mediate effects on cellular concentrations of thymidine-(analogue) anabolites. The absence of mtDNA depletion associated with haplotype 1 may result from greater phosphorylation of AZT-MP, resulting in decreased cellular AZT-MP concentrations and increased AZT-TP concentrations. Sommadossi and colleagues 317 demonstrated that although equivalent amounts of AZT-TP and d4T-TP are incorporated into bone marrow DNA, AZT-TP fails to be excised due to the high cellular concentrations of the MP, which is known to inhibit the 3’-5’ exonuclease activity of DNA polymerases and proposed this as a mechanism for the selective bone marrow toxicity of AZT. Thus, haplotype 1 may be associated with greater excision of AZT-
TP from incorporated DNA than other haplotypes and thus less mtDNA depletion.
However, the observed preservation of mtDNA levels associated with haplotype 1 in this study occurred in adipocytes, where d4T, which does not accumulated in the MP form, consistently causes greater mtDNA depletion than AZT. Thus, haplotype 1 may mediate less mtDNA depletion simply through less AZT-MP phosphorylation and thus lower AZT-TP concentrations and therefore less AZT-TP incorporation.
This is consistent with the suboptimal intracellular nucleotide pool mechanism of
HIV-mediated haemotoxicity proposed by Bofill and colleagues 363 . If haplotype 1
mediates less mtDNA depletion than the other haplotypes through less AZT-MP
phosphorylation, haplotype 3 may mediate higher pre-treatment haemoglobin
concentrations through greater thymidine phosphorylation. In rapidly dividing cells
136 such as CD34 + haematopoietic progenitor cells, which differentiate into both myeloid and erythroid lineages and have been shown to support infection of both M- tropic and T-tropic HIV-1364-366 , an inability to maintain optimal nucleotide pool composition in the presence of HIV-1- infection would make it impossible for progenitor cells to complete the cell cycle and terminally differentiate 363 . Thus haplotype 3 may maintain optimal dT-TP concentrations in the presence of untreated
HIV-1 infection. However, conflicting data exists regarding the susceptibility of
CD34 + cells to HIV-1 infection in vitro 367-370 and in vivo 371,372 .
In conclusion, although the mechanism by which dTMPK genetic variation influences pre-treatment haemoglobin concentrations and adipocyte mtDNA values remains to be determined, these data suggest that dTMPK haplotyping may have promising applications in identifying individuals relatively protected from developing lipoatrophy in response to AZT therapy and may play a role in identifying individuals susceptible to anaemia in susceptible populations.
Pharmacogenetics has proved to be clinically relevant in predicting neuropsychological toxicity associated with efavirenz 373 and hypersensitivity to abacavir 374,375 , and this study suggests that pharmacogenetics may prove useful in predicting AZT toxicities.
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