Clinical Importance of DNA and RNA determinants

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1 Clinical Importance of DNA and RNA determinants Akin Abayomi MD Lady Meade Ref Unit Lab Barbados and Division of Haematopathology, University of Stellenbosch, Cape Town, South Africa.

2 CCAS Not Terrorist but Arsonist Lit the fire Right amount of fuel. Caribbean was ready. Success has been dependent on CDC and CHART right from the beginning.

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4 Good fires Good fires are ecologically crucial, clearing out dead brush and returning nutrients to the soil. Trees respond to fire like roses respond to pruning and fertilizer.

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6 Content HIV infection, integration and latency Effective controllers vs Progessors and set point. HIV pathogenesis (IA) Immune activation. VL is directly proportional to IA Laboratory determinants DNA and RNA Safe Blood Infant Diagnosis HEU Viral Load Viral set point and risk of infectivity Treatment monitoring Acute infection Test and Treat DART VL and escape Mutations, fitness

7 HIV Gaps We don t quite understand this disease yet Much of what I say is still speculation Evidence based speculation! Goal post changing all the time

8 Unless we hold a gun to the HIV brain, we may never know the truth. HIV is Cat with 9 lives Knows more immunology than Prof Janossy and Lydyard put together.

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12 Koch s Postulates The micro-organism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy persons. Her in lies the problem in HIV: Expensive Dangerous Confusing

13 Proof of infection Identify the micro-organism Culture Look for parts (some genetic material) Look for the immune response to the infection eg Antibodies or CMI Find typical features associated with the infection

14 In any fatal Clonal Disease When or who to treat Response to treatment When to stop treatment Risk of relapse on or off treatment

15 Molecular Biology is all about Jeans Viral genes Host genes

16 Human Genetic composition 22 pairs + 1 pair sex-determining chromosomes one chromosome of each pair donated from each parent s egg or sperm sex chromosomes: X,Y for males; X,X for females Human Genome Project Information Website

17 Genomic Molecular Biology 101 Principles of DNA transcription mrna translation and gene expression Stability of genomes

18 Genome Facts Nucleotides chromosomes genes base pairs A-T, C-G comprised of nucleotides adenine thymine guanine cytosine

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21 DNA Double Helix and Specificity Each strand comprises of nucleotides binds to its complementary DNA strand. This unique property of the DNA structure allows stable custody of genetic material (fidelity). We make use of that fidelity to make true copies of genes for analysis.

22 Gene Facts size of human genome: 3 billion base pairs (bp) number of human genes: ~100,000 genes vary in length and can cover thousands of bases avg. size: ~3,000 bp only about 5% of the human genome contains genes function of much of the genome is unknown

23 Gene Expression RNA Transcription Protein Translation

24 DNA to RNA Deoxyribose nucleic acid is the master code (Nucleus) DNA (C:G and A:T) Transcribed into Ribose Nucleic acid (ctytoplasm) RNA (C:G and U:T) RNA translated into proteins for biological function

25 HIV Is a retrovirus Unlike humans keep its genetic information in the form of RNA RNA must be converted to DNA Moves in and will overwhelm cell Uses cell machinery to replicate Viral genetic material indicative of infection

26 HIV Immunobiology HIV attachment to cell membrane of host Injection of HIV RNA and proteins RT of HIV converts RNA to a cdna Integration of cdna into human genome Transcription in activated cells Death of infected cell (EM) Latency in un activated cell (Naïve or CM)

27 Integration Stable HIV cdna integration In at least 400 active human gene sites Uses cell machinery to produce daughter viruses Rate of replication is dependent on cell activation (NFkB Status)

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29 Options for detecting HIV genetic material Integrated HIV DNA or cdna Transcribed HIV RNA from integrated cdna template

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32 Detectable viral NA (DNA or RNA) Detecting viral NA helps one to read the latest update on what the virus is doing Amount type and location is probably a reflection of burden of disease controlled by: Immune control Host factors Viral fitness Level of Immune activation ART impact

33 Type and location HIV NA in plasma (RNA) Plasma HIV NA in CD4 cells (DNA) Nucleus Activated CD4 cells (CCR5+) Quiescent CD4 cells ( CCR5- Reservoirs)

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35 Visualizing genes Identify it with primers Make plenty of it PCR, Branched DNA, Nuclisense Stain or mark it with a probe for visualization Is the correct product present?

36 Nature of the test Is it there or not? Simple PCR(Qualitative) How much is there? (Quantitative) Q-PCR

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39 Simple PCR Indicates if a specific sequence is present or not. Question: Is there a copy of HIV cdna in CD4 cells of patient? Indicative and confirmation of infection Useful for definitive diagnosis especially where HIV abs may be ambiguous or absent Neonatal diagnosis Vaccination trials

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41 Uses Early Infant Diagnosis Confirm a diagnosis in a pt on vaccination trial

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44 Polymerase Chain Reaction -PCR- Specificity of DNA hybridization: use primers to surround the target DNA sequence In vitro synthesis of the target DNA sequence Multiple cycles to exponentially increase the amount of the target DNA sequence Detection of the amplified target DNA sequence

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47 Sample Extraction

48 Detection of product Visually on a electrophoretic gel Absorbance on a photometer

49 PCR Temp optimization Lower temperatures Increased yield Decreased specificity Higher temperatures Increased specificity Decreased yield Optimal temperature Maximum yield with Maximum specificity

50 PCR - photometer Photometer Detection - Absorbance is read at 660nm in the photometer. Compute r Printer

51 Viral Load So what is this Viral Load? Plasma Viral Load Integrated Viral Load More expensive than simple PCR! Requires more infrastructure and expertise. Many platforms now Taqman, Nuclisense, Branched DNA and NAAT

52 HIV QUANTITATIVE PCR or Viral Load Knowing the initial amount of target. A measure of the disease burden compartment. Viral mrna as well as integrated cdna are amenable to this test

53 Plasma pvl and Integrated ivl Quantitative estimates of HIV turn over Number of host cells actively infected (active and latent)

54 pvl Amount of free HIV RNA in plasma Expressed as copies per ml of plasma Standard >400 c/ml Sensitive >50 c/ml Ultra sensitive 3-50 c/ml

55 ivl Amount of cdna found in a given number of lymphocytre popultation Expressed as DNA copies per 10,000 cells Reflection of the number of cells infected In pt with undetectable pvl gives indication of reservoir.

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60 Real-Time RT-PCR Compare the data in the exponential phase instead of at the end point of the PCR reaction where all the samples reach the same N

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62 A Calibration Curve for an Absolute Quantitation

63 Stages of infection and use of HIV NA detection Acute PHI Blood donors to narrow down window (pvl) Early childhood detection (cdna PCR or pvl) Vaccination trial (cdna) Well controlled Infectivity (pvl) When to start (pvl debatable value) Effect of ART (pvl) Latency (ivl)

64 Blood donors (pvl) Window period NAAT (pvl) is positive before antibodies are detectable Pooling can save costs and retain power of early detection

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66 Early Detection in Minipools 16 of donated blood prevents infection in 1 in every 2 million transfusions NEJM :760

67 Paediatric Early detection (PCR or pvl) Children born to HIV+ mothers Passive antibodies are transferred to fetus Placenta Breast Detection can only be confirmed with NA based test looking for HIV cdna or pvl or ivl.

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69 Value of early Diagnosis in children Why would we want to make an early diagnosis?: Psychological Prevention and infant feeding strategies Early Rx benefits

70 HIV Exposed Uninfected infants (HEU) Mother pos, Baby Negative. Evidence suggests they still have an immune aberration (IAS abstract 2009) Should be watched as closely as infected babies SA cohort indicates high incidence of PCP, Pneumonia and meningitis

71 Patient with HIV When to start! Test and Treat? Cost of Rx, Side effects, Immune reconstitution How to measure impact of treatment? pvl, CD4 and resistance mutations Expensive Dart study suggest monitoring is too expensive pvl may be stable but resistance may be evolving When to change?

72 Why Measure Viral Load? Combination antiretroviral therapy that suppresses HIV replication to undetectable levels can delay or prevent the emergence of drug-resistant viral variants

73 pvl Current treatment strategies suggest suppress viral activity to <50 c/ml Other factors are also important Suppressing immune activation Immune reconstitution Depleting the latent reservoirs

74 HIV-1 Testing Limits of Detection Roche Monitor Standard Roche Monitor Ultrasensitive Bayer Quantiplex HIV-1 RNA 3.0 Organon-Teknika NucliSens HIV-1 QT 400 copies/ml 50 copies/ml 50 copies/ml 80 copies/ input

75 Patient Management Initiation of therapy Viral load, CD4 cell count, symptoms Combination therapy - HAART Monitoring response to therapy Viral load, lack of response, rebound Treatment failure Resistance testing

76 Important implications of VL Infectivity Relationship with IA Resistance testing Relationship with co infection Vertical transmission Latency

77 Cell Associated VL Hockett JEM 1999 Irrespective of VL mean HIV RNA copy number per infected cell is 3.6 log10 copies. Number of infected lymph node CD4 cells is proportional to pvl. Residual cells expressing this same mean copy number are detectable (frequency <2/106 cells) in tissues of Rx patients who have undetectable pvl (<50 copies/ml).

78 Relationship of plasma viral load to HIV vrna expressing cells in lymph node tissue Hockett et al. Journal of Experimental Medicine 2009:189:

79 Mean HIV RNA VIRADAPT: Mean change in HIV-1 RNA from baseline Randomized Study Open Study Control Genotype Time, month Clevenbergh. Antiviral Ther. 1999;4(suppl 1):42., Durant. Lancet 1999;353:2195.

80 Infectivity New evidence suggest infection is effected from cell to cell interaction or the infective synapse

81 Function of infectivity The amount of free plasma mrna is a predictor of infectivity. Reflects number of infected cells in the genital and mucosal membranes

82 Uganda discordant couple study Quinn NEJM pvl significantly higher pos partners who infected neg partners (90,254 vs. 38,029 c/ml. No transmission when pvl of less than 1500c/ml Each log increment in the pvl inceases risk of seroconversion by a 2.5 factor No seroconversions among the 50 circumcised male partners irrespective of VL

83 Heterosexual & Vertical Transmission Benki JCM 2006 ivl copy number in endocervical secretions from women with low pvl who were non transmitters was 1.8 copies/10,000 cells. Women with higher pvl had 16.6 copies/10,000 cells Data suggest genital HIV cell-associated virus load determines infectivity.

84 Impact of Co-infections So for us in Africa and some parts of the Caribbean Co infection increases chance of infectivity Well characterized in case of Malaria

85 Malaria HIV interaction 500 million cases and 1 million deaths from malaria per year 40 million cases and 1 million deaths from HIV per year Dramatic overlap of disease bounderies Must be some interaction

86 Malaria HIV HIV pt with clinical malaria have a 1 log increase in pvl which lasts for up to 6 months Increases risk of transmission by 2.5

87 Malaria and HIV (Uganda study) Kisumu district population of 200,000 HIV cases total 50,000. Absence of malaria intervention may account for a cumulative 8,500 excess HIV infections 1 million excess malaria episodes since 1980 because of HIV induced susceptibility.

88 Factors determining set point Determined by many host and viral factors. Immune activation state at the time of PHI, modality of infection, co infections Host phenotype eg, CCR5 and HLA constitution Robustness of innate CM and humoral resposes.

89 Elite controllers, Slow Progressors and progressors Elite controllers" 1% of the HIV-infected population. Undetectable levels of pvl <50c/ml and stable CD4. Long-term nonprogressors, detectable pvl >50c/ml but maintaining a CD4+ count of >500. Progressors: Varriably high pvl and declining CD4

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91 Fig. 4. Potential effect of vaccination on HIV load B. D. Walker et al., Science 320, (2008) Published by AAAS

92 Surround myself with great minds to name a few Clive Songee Nami Kelly George Frank Maurice Chris Brendan Maria CCAS members CHART members CDC members Local organizing Com Cynthia Warner

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102 Time to think out of the box

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106 Fig. 1. The time course of HIV and malaria interaction in Kisumu, Kenya L. J. Abu-Raddad et al., Science 314, (2006) Published by AAAS

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108 Phase 2. Latency or chronic infection

109 Fraser PNAS 2007 We quantify the transmission potential as a function of set-point viral load and find that it is maximized for intermediate viral loads, which we observe are also the most common among untreated patients. Although individuals with high viral loads are the most infectious in the short term, the total contribution to infection of those with intermediate viral loads is found to be larger because of the longer duration of asymptomatic infection. this leads us to hypothesize that HIV-1 could have evolved to optimize its transmissibility, a form of adaptation to the human host population The consequences for public health and an evolutionary hypothesis arising from this observation are discussed.

110 The distribution of set-point viral loads Fraser C et al. PNAS 2007;104: by National Academy of Sciences

111 Set-point viral load and duration of asymptomatic infection Fraser C et al. PNAS 2007;104: by National Academy of Sciences

112 Set-point viral load and infectiousness Fraser C et al. PNAS 2007;104: by National Academy of Sciences

113 Transmission potential Fraser C et al. PNAS 2007;104: by National Academy of Sciences

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115 Effect of ART

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117 Relationship between IA and VL

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123 Relative risk of Dying based on CD4 count

124 Starting ART at PHI?

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126 When to start

127 Future strategies to restore immune function in HAART controlled viral suppressed pts

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130 II.2 Pros/Cons PCR DNA PCR RNA PCR Ultra-sensitive p24 A D V A T A G Highly sensitive and specific for HIV validated and widely used for infant diagnosis gold standard for early infant diagnosis Validated for DBS Highly sensitive and specific for HIV Provides additional virological information for staging disease Can be run on VL Multiple RNA PCR assays commercially available Reagents are less costly ELISA based technology E S C H A L L E N G E S Costly Technologically complex Labor intensive Assay registered with FDA for Research Use Only Kits need cold chain (4 o Celsius) Only 1 DNA PCR assay, which is from Roche, is commercially available More costly than DNA PCR Technologically complex Labor intensive Kits need cold chain (4 o Celsius) Not validated for DBS sample Mainly used for research purposes Not widely available Small time window of detection Sensitivity not verified Equipment and consumables provided by many different suppliers, making procurement difficult Not validated for DBS

131 MINISTRY OF HEALTH KENYA ALGORITHM FOR EARLY INFANT DIAGNOSIS FOR HIV EXPOSED CHILDREN WELL CHILD SICK CHILD (Manage presenting illness and stabilize) START COTRIMOXAZOLE PROPHYLAXIS, AB TEST TO INFANT IF EXPOSURE NOT KNOWN 6 WEEKS DBS (PCR) If <18 months DBS (PCR*) HIV- HIV- HIV+ Evaluate for ART start on ARV if Eligible Counsel for exclusive BF and early weaning at 6 months Antibody testing 9 and 12m HIV+ Evaluate for ART start on ARV if Eligible Continue child welfare services. If still breastfeeding counsel for exclusive BF and early weaning at 6 months Antibody testing 9 and 12m If HIV+ at 12 m If HIV-ve stop CTX, if not B/F for at least 3 Months If HIV+ at 12m If HIV-ve stop CTX, if not B/F for at least 3 Months Evaluate for ART start on ARV if Eligible Confirmatory AB test at 18m Evaluate for ART start on ARV if Eligible Confirmatory AB test at 18m FOR MORE INFORMATION CONTACT THE NATIONAL AIDS/STD CONTROL PROGRAMME (NASCOP) P.O. BOX NAIROBI TEL: FAX

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133 Dried Blood Spot (DBS) sample collection 1. Collection site 2. Collection 3. Drying 4. Packaging (silica desiccant packs, and humidity indicator cards)

134 Carr 2007 Measurement of cvl and ivl has the potential to be of value for patients with undetectable pvl (<400 or <50 copies/ml), where additional data reflecting ongoing viral replication could assist our understanding of progression of infection. For patients with undetectable pvl and for whom cvl (n = 37) and ivl (n = 26) were quantitated, pvl and CD4 counts were monitored for the subsequent 12 months. During this time therapy for four patients failed as measured by increased pvl (Fig. 2B and C; Table 3). Elevated cvl (average value, >2,000 copies/105 cells [Table 4]) was not associated with therapy failure with broad cvl for patients failing therapy, and only one out of nine patients with cvl of >2,000 copies/105 cells failing therapy in the subsequent 12 months. All four patients for whom therapy failed had higher than average ivl values (>27 copies/105 cells [average values are shown in Table 4]), including the patient with the highest ivl observed. Seven out of eleven patients with ivl of >27 copies/105 cells and 15/15 patients with ivl of <20 copies/105 cells were maintained on suppressive therapy, suggesting that patients with ivl of <20 copies/105 cells were less likely to fail therapy within the next 12 months than patients with higher ivl values. However, more patient numbers are needed to substantiate any prognostic value of ivl measurements and determine critical values above which therapy failure becomes probable.

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136 Butler 2002 Following introduction of the viral core into the cell cytoplasm, the viral RNA is reverse transcribed to yield a linear cdna copy (Fig. 1). In productive infections, the viral cdna is then integrated into a chromosome of the host cell. A fraction of the linear cdna molecules instead becomes circularized, a reaction that is thought to be a dead end for the virus since circular cdnas are not substrates for the viral integration machinery (3). Circularization can be accomplished by (i) ligation of the cdna ends by the host cell nonhomologous DNA end-joining system (9), yielding the two-long terminal repeat (2-LTR) circle; (ii) recombination between the two LTRs, yielding a 1-LTR circle; (iii) stalling of reverse transcription to yield 1-LTR circles; or (iv) integration of the linear cdna into itself, yielding an internally rearranged form.

137 Vatakis 2009 JV The identification of HIV integration sites was determined by an inverse PCR assay (Fig. 1A) (19, 41). Based on the sequencing analysis, we identified a total of 451 integration site sequences in quiescent cells (G0) (Table 1). Furthermore, we found 16 sites self-integrated into the provirus genome, LTR circles, and 109 sites that were classified as unidentified because they contained sequences corresponding to the junction of the left LTR and primer binding site, which can be derived from 1-LTR, 2-LTR or linear viral DNA (integrated and unintegrated) (Table 1). In the stimulated cell group (Stim), we identified 543 integration sites, 38 autointegrations, and LTR circles and isolated 190 unidentified sites

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139 Vatakis Based on the above-described data, we can propose a model of HIV integration in quiescent and stimulated CD4+ T cells (Fig. 5C). While site selections are quite similar between the two cell types, the process in quiescent cells is riddled with processing errors, which do occur in stimulated cells but are exaggerated in quiescent T cells. These errors result in the formation of LTR circles, many of them abnormal. This may partially explain the lack of protein expression in quiescent T cells and the poor rescue observed when they are immediately stimulated after infection

140 Intensification does not reduce residual viremia in patients on ART Dinoso J B et al. PNAS 2009;106: by National Academy of Sciences

141 Dinoso PNAS 2009 Here, we show that intensification of ART does not reduce residual viremia in patients with HIV-1 RNA levels <50 copies per milliliter. This finding is consistent with qualitative studies showing that the residual virus is genetically stable and lacks new resistance mutations (24 26) with quantitative studies suggesting that the level of residual viremia is a reflection of the size of stable compartments established before therapy (18, 19) and with observations of consistently low levels of residual viremia in patients on different ART regimens (18). The absence of further decline in viremia with antiretroviral intensification studies supports our thesis that standard combination therapy is maximally suppressive. Because our findings are consistent with the hypothesis that residual viremia primarily represents output from stable reservoirs of infection, strategies for identifying and targeting these reservoirs may be the next important step in eliminating the persistence of HIV-1 infection.

142 Sanctuary Sites CNS The central nervous system (36, 37) (CNS) and genitourinary (GU) tract (38) are regarded as distinct compartments of HIV-1 infection in which antiretroviral penetration may be reduced (39 45). The recent CHARTER study (46) measured levels of ATV in cerebrospinal fluid (CSF); even with RTV boosting, CSF levels were 100-fold lower than plasma levels and were lower than the reported ATV IC50. Additional studies from this group (45) have rank-ordered penetration of antiretrovirals into CSF; some older drugs (e.g., indinavir) have higher relative penetration than newer agents, such as ATV and tenofovir. With regard to the drugs used for intensification in our study, LPV/r was considered to have a high CNSpenetration effectiveness score (45). Based on ratios of penetration into sanctuary sites and clinically determined trough plasma concentrations (BMS EFV package insert), we estimate EFV levels in CSF and semen to be 1.8-fold and 30-fold higher, respectively, than the IC90 (6 ng/ml) (31). Thus, these 2 drugs may give better CNS penetration than ATV/r. It is also important to note that the real penetration of any antiretroviral into the brain has not been extensively studied, and studies using CSF as a surrogate show that CSF may not accurately reflect levels of virus or drug penetration in brain parenchyma (47). In addition, NNRTIs and PIs are strongly protein-bound, and the reduction in drug levels in compartments such as CSF may be partially compensated by the substantial reduction in protein concentration in this site (48

143 Sanctuary Sites GU With regard to the GU tract, previous studies have shown that individuals on EFV-based ART have undetectable (<5 copies per milliliter) HIV-1 RNA levels in semen while maintaining low but measurable levels (between 5 and 50 copies per milliliter) in plasma (16).

144 Viremia source in fully Rx pt results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.

145 Summary Sanctuary sites Collectively, available evidence suggests that detectable HIV-1 RNA in the CNS and GU compartments during ART is uncommon and that viral replication in these compartments is unlikely to make a major contribution to residual viremia in most treated patients.

146 Latency principle Duverger JV 2009 Recent research has emphasized the notion that human immunodeficiency virus type 1 (HIV-1) latency is controlled by a restrictive histone code at, or DNA methylation of, the integrated viral promoter (long terminal repeat [LTR]). The present concept of HIV-1 latency has essentially been patterned from the principles of cellular gene regulation. Here we introduce an experimental system that allows for the qualitative and quantitative kinetic study of latency establishment and maintenance at the population level. In this system, we find no evidence that HIV-1 latency establishment is the consequence of downregulation of initial active infection followed by the establishment of a restrictive histone code at the viral LTR. Latent infection was established following integration of the virus in the absence of viral gene expression (silent integration) and was a function of the NF- B activation level in the host cell at the time of infection. In the absence of a role for epigenetic regulation, we demonstrate that transcriptional interference, a mechanism that has recently been suggested to add to the stabilization of HIV-1 latency, is the primary mechanism to govern latency maintenance. These findings provide direct experimental evidence that the high number of viral integration events (>90%) found in actively expressed genes of CD4+ memory T cells from highly active antiretroviral therapy-suppressed patients represent indeed latent infection events and that transcriptional interference may be the primary mechanism to control HIV-1 latency in vivo. HIV-1 latency may thus not be governed by the principles of cellular gene regulation, and therapeutic strategies to deplete the pool of latently HIV-1-infected cells should be reconsidered.

147 Eradicating latency cure Hung- Chih Yang PNAS 2009 HAART) can suppress plasma viral load to undetectable levels, viremia rebounds within weeks after discontinuation of HAART. The major barrier to eradication of HIV-1 infection is the existence of viral reservoirs. Among them, the best characterized is a small pool of latently-infected resting memory CD4+ T cells harboring an integrated provirus (2 4). Previous studies have demonstrated the stability of this latent reservoir in patients on HAART (5). The half-life of this reservoir was estimated to be >44 months. At this rate of decay, it is expected to take >60 years to purge HIV-1 from infected patients on HAART. Thus, this reservoir necessitates the lifetime use of HAART, and strategies are needed for eradication of latently infected cells

148 Hung-Chih Yang PNAS 2009 Recently, reactivation of latent virus has gained wide interest as a potential strategy to eradicate the viral reservoirs (8 11). It is assumed that latently infected cells can be killed either by immune attack or direct viral cytopathic effects after reactivation of latent HIV-1. A reactivation strategy, along with simultaneous efficient suppression of viral spread by HAART, might reduce and ultimately eliminate the latent reservoirs (6, 7).

149 Activation Options (Berlin Patients) Allo transplants with Deltta 32 CCR5 homologous phenotype results in undetectable VL after recovery. Indicating that lymphocytes are the important source of latency

150 Activation Options (LTR) Hung- Chih Yang PNAS 2009 the host transcription factor NF-κB is important for activating HIV-1 gene expression through 2 conserved κb sites in the core enhancer region of the HIV-1 LTR (12, 13). However, HIV-1 can replicate in the absence of κb sites in the LTR (16), consistent with the existence of NF-κB-independent pathways in the activation of HIV-1 (17, 18). NF-κB also has a critical role in innate and adaptive immune responses, and regulates genes that have important roles during T cell activation (19). Because of the central role of NF-κB in T cell activation, we reasoned that to find genes that could uncouple the activation of latent HIV-1 from T cell activation it would be desirable to identify factors that could activate the HIV-1 LTR in an NF-κB-independent manner.

151 Delta 7 ETS-1 Hung-Chih Yang PNAS 2009 Full-length Ets-1 has previously been shown to participate in LTR regulation in concert with other transcription factors, like NF-κB, NFAT, and USF-1 (26, 27), by binding to a conserved Ets-1-binding site ( 150 to 145) in the distal region of the LTR (32). Our results demonstrate that overexpression of the alternatively spliced form of Ets-1, ΔVII-Ets-1, is sufficient both for LTR transcriptional activation and activation of latent HIV-1.

152 Hung-Chih Yang PNAS 2009 The ability of ΔVII-Ets-1 to reactivate latent HIV-1 suggests that potential therapies might be aimed at increasing the levels of expression of this isoform of Ets-1 in latently infected cells. Alternatively, potential therapies might involve the conversion of fl-ets-1 from an inhibited to an active transcription factor.

153 Berlin Patient Hutter NEJM 2009 Infection with the human immunodeficiency virus type 1 (HIV-1) requires the presence of a CD4 receptor and a chemokine receptor, principally chemokine receptor 5 (CCR5). Homozygosity for a 32-bp deletion in the CCR5 allele provides resistance against HIV-1 acquisition. We transplanted stem cells from a donor who was homozygous for CCR5 delta32 in a patient with acute myeloid leukemia and HIV-1 infection. The patient remained without viral rebound 20 months after transplantation and discontinuation of antiretroviral therapy. This outcome demonstrates the critical role CCR5 plays in maintaining HIV-1 infection.

154 Clinical Course and HIV-1 Viremia Hutter G et al. N Engl J Med 2009;360:

155 Phase 1: Acute Infection

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