VH1 latente proporciona pistas para la erradicación de depósitos a largo plazo

The HIV‑1 pandemic represents one of the great plagues in human history and a major challenge for medicine, public health and biological research. Infection with HIV‑1, which was first isolated in 1983 (REF. 1), causes AIDS, a syndrome that was first described in 1981 (REF. 2). Continuous research has allowed the devel‑opment of effective treatments that have transformed HIV‑1 infection from a fatal illness into a chronic dis‑ease3. Currently, 25 different active compounds belong‑ing to 6 different drug families have been developed. However, regardless of the use of highly active antiretroviral therapy (HAART), a cure is not yet achievable, and HIV‑1 persistence in reservoirs represents the major obstacle for its eradication4,5.

All lentiviruses can infect macrophage lineage cells, in which they generate a persistent infection6. HIV‑1 is a lenti virus that has developed a broader tropism, leading to preferential infection of CD4+ T cells, which are severely destroyed during the illness7. This target provides two potential environments for the virus, allowing latency in resting CD4+ T cells and massive viral replication in activated cells8.

The molecular mechanisms that lead to HIV‑1 reac‑tivation have been characterized in detail, but the study of viral latency remains limited. Recently, the identifi‑cation of factors that restrict retroviral infections9, the characterization of chromatin structure in the setting of viral integration10, the discovery of new systems that regulate gene expression (such as small interfering RNAs (siRNAs)11) and the development of new techniques for analysing HIV‑1 latency12 have provided a new

perspective on this concealed state. Indeed, latency should not be considered a merely passive event but, rather, an active process that is maintained by cellular elements. In this Review, the molecular mechanisms that are necessary for the establishment of HIV‑1 latency, their relationship with different cellular and anatomical reservoirs and the current treatment strategies to target viral persistence are analysed.

HIV‑1 life cycleThe life cycle of HIV‑1 can be divided into two phases: the early stage occurs between entry into the host cell and integration into its genome, and the late phase occurs from the state of integrated provirus to full viral replication. Accordingly, two types of viral latency can be differentiated: pre‑integration latency refers to the gen‑eration of different forms of viral DNA before integra‑tion, whereas post‑integration latency refers to the lack of replication after the insertion of viral DNA into the host genome8,13. Viral entry involves the fusion of viral and cellular membranes through successive interactions with CD4 and CXC chemokine receptor type 4 (CXCR4) or CC chemokine receptor type 5 (CCR5)7 (FIG. 1). The HIV‑1 core, containing two single‑stranded, plus‑sense RNA genomes, tRNA primers, viral protease, retro‑transcriptase and integrase, is released into the cytosol. This intracellular core is named the reverse transcrip‑tion complex because most of the viral RNA genome is reverse transcribed by the retrotranscriptase to form a linear, mostly double‑stranded cDNA (dscDNA) molecule with terminal direct repeats and blunt ends14.

AIDS Immunopathology Unit, National Centre of Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain.e‑mails: [email protected]; [email protected]; [email protected]; [email protected]:10.1038/nrmicro2223

Highly active antiretroviral therapyA combination of three or more potent anti-HIV-1 drugs that reduces viral load below detection limits by standard techniques.

ReservoirA cell type or anatomical site in which a replication-competent virus persists for much longer than it does in the main pool of productive infected cells, thereby sustaining the infection. Integrated proviral genomes and cells that persistently replicate HIV-1 in the presence of highly active antiretroviral therapy can be considered viral reservoirs.

Understanding HIV‑1 latency provides clues for the eradication of long‑term reservoirsMayte Coiras, María Rosa López‑Huertas, Mayte Pérez‑Olmeda and José Alcamí

Abstract | HIV‑1 can infect both activated and resting, non‑dividing cells, following which the viral genome can be permanently integrated into a host cell chromosome. Latent HIV‑1 reservoirs are established early during primary infection and constitute a major barrier to eradication, even in the presence of highly active antiretroviral therapy. This Review analyses the molecular mechanisms that are necessary for the establishment of HIV‑1 latency and their relationships with different cellular and anatomical reservoirs, and discusses the current treatment strategies for targeting viral persistence in reservoirs, their main limitations and future perspectives.

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Nature Reviews | Microbiology

Budding and maturation

Genomic RNA

Translation ofviral proteins

Full- and short-length dscDNA

Microtubulenetworkand actinfilaments

LineardscDNA

Circular, non-integratedDNA

Pre-integrationlatency

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Integration

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Reversetranscriptioncomplex

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UnsplicedmRNA

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Rev

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NPCProviralDNA synthesis

Assembly

NF-κB,SP1, NFAT,Tat

ProvirusViral genomic double-stranded cDNA that has permanently integrated into the host cell genome and that acts as a template for the synthesis of viral RNAs.

Direct repeatsTwo or more identical or nearly identical repeats of specific nucleotide sequences that are in the same direction in the DNA molecule.

Blunt endThe end of a double-stranded DNA molecule that terminates in paired bases, rather than with uneven ends, such that one strand overhangs.

Enhancer elementA DNA consensus site, usually located 5′ from the basal gene promoter, that is bound to by specific transcription factors to increase the rate of transcription of the gene that it controls. An enhancer element may be placed thousands of bases upstream or downstream of the transcription initiation site of this gene.

once in the cytoplasm, the reverse transcriptase complex progressively disassembles to form the pre‑integration complex (PIC), which is composed of linear dscDNA, integrase, matrix protein, retrotranscriptase, viral protein r (Vpr) and various hosts proteins, such as the high‑mobility group protein b1 (HmG1) or the lens epithelium‑derived growth factor (leDGF; also known as PSIP1)15–18. Viral trafficking is mediated through microtubules by retrograde transport and uses dynein to move towards the nuclear pore complex19. At the nuclear pore complex, the PIC enables the transport of dscDNA through the intact nuclear envelope, thereby allowing the infection of resting, non‑dividing cells. linear dscDNA either integrates into the host cell chromosomes or circularizes as one or two long terminal repeat (lTR)‑containing circles16. Host factors such as emerin and

leDGF are key molecules that facilitate HIV‑1 integra‑tion20,21. emerin is an inner nuclear envelope protein that seems to increase the efficiency of viral integration in macrophages by improving the localization of viral DNA to the chromatin before integration22, whereas leDGF is a transcriptional co‑activator that binds integrase and acts as a tethering factor to promote viral integration18. After integration, the lTR‑flanked provirus behaves as a cellular gene: the 5′ lTR operates like any eukaryotic promoter and the 3′ lTR acts as the polyadenylation and termination site23.

Activation of the T cell induces binding of the tran‑scriptional pre‑initiation complex to enhancer elements in the 5′ lTR proximal promoter (FIG. 1). This complex gathers essential host transcription factors, such as nuclear factor‑κb (NF‑κb)24, nuclear factor of activated

Figure 1 | HIV‑1 life cycle and viral latency. Viral fusion and entry requires the binding of glycoprotein gp120 to CD4 receptors at the cell surface as well as to CC chemokine receptor type 5 (CCR5) or CXC chemokine receptor type 4 (CXCR4). The viral nucleocapsid enters the cytoplasm and uses cytoplasmic dynein to move toward the nuclear pore complex (NPC). The viral RNA is retrotranscribed into proviral double‑stranded cDNA (dscDNA), which can stay in the cytosol, where it is highly unstable and exists in a transient, reversible pre‑integration latent state, or can form a pre‑integration complex consisting of dscDNA, viral proteins and some host cell proteins. When ATP levels are adequate, the pre‑integration complex is transported into the nucleus through the NPC, and the dscDNA either circularizes as one or two long terminal repeat‑containing circles or is integrated into a host cell chromosome. After integration, the provirus remains quiescent, existing in a permanent post‑integration latent state. On activation, the viral genome is transcribed by the synergic interaction of cellular transcription factors (nuclear factor‑κB (NF‑κB), nuclear factor of activated T cells (NFAT) and specificity protein 1 ( SP1)) and the viral transactivator, Tat. Rev, a viral protein, regulates the splicing and cytosolic transport of some of the viral mRNAs, which are translated into regulatory and structural viral proteins. New virions assemble and bud through the cell membrane, maturing through the activity of the viral protease.

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TatA regulatory HIV-1 protein that is essential for viral transcript elongation through its interaction with the Tat response element and several host factors, such as the positive transcription elongation factor b.

RevA regulatory HIV-1 protein that controls the nuclear export of viral mRNA species through its interaction with the Rev response element that is found in unspliced or incompletly spliced HIV-1 RNAs.

Naive T cellA mature T cell from the acquired immune system that has not yet made contact with its cognate antigen and that therefore lacks both activation and memory markers on the cell surface.

Memory T cellA T cell that persists for a long time after its exposure to a specific foreign antigen and that can be promptly expanded to effector T cells after contact with the same antigen to initiate a faster and stronger immune response.

Retroviral restriction factorA component of the innate immune system that aids the survival of a host cell after retroviral infection by interfering with viral replication at different steps of the viral life cycle.

T cells (NFAT)25 and specificity protein 1 (SP1)26. These enhancer proteins transmit activation signals to basal factors belonging to the general transcription machinery and promote binding of RNA polymerase II (RNAPII) to the TATA box to initiate mRNA transcrip‑tion. A 59‑nucleotide stem‑loop structure termed the transactivation response element (TAR) is then formed at the 5′ end of the nascent viral transcript, creating a binding site for the viral transactivator Tat27. The Tat–TAR interaction promotes efficient elongation of viral transcripts by recruiting cellular factors that increase the functional capacity of RNAPII, such as positive transcription elongation factor b (PTeFb), which is composed of cyclin‑dependent kinase 9 (CDK9) and cyclin T1 (REFs 28,29). efficient elongation of viral tran‑scripts allows the synthesis of mRNA, which is further processed by the regulatory protein Rev. Rev is a viral RNA‑binding factor that regulates the nucleo‑cytosolic transport and splicing of viral mRNA species30. once in the cytoplasm, HIV‑1 proteins are synthesized as large precursor polyproteins that must be processed into mature viral proteins and then assemble to create viable particles that will bud off the cell and infect new cells31 (FIG. 1).

Cellular environment and HIV‑1 replicationResting lymphocytes: the territory of latency. The acti‑vation of resting naive T cells gives rise to effector T cells (BOX 1). most of these die during the immune response, but some return to a resting state and become memory T cells (FIG. 2a). both naive and memory subpopula‑tions of resting lymphocytes represent an extremely restrictive environment for HIV‑1 replication, owing to several mechanisms. First, CCR5 expression is low or absent, which limits the number of targets available to the virus32, and second, low nucleotide pools and ATP levels in these cells cause inefficient retrotranscrip‑tion33,34 and limited nuclear PIC import, respectively35. moreover, the virus must overcome several cellular retroviral restriction factors, such as the cytoplasmic APobeC3G (apolipoprotein b mRNA‑editing enzyme catalytic polypeptide‑like 3G). This cytidine deami‑nase exists in resting CD4+ T cells as an enzymatically active, low‑molecular‑mass complex and interferes with reverse transcription through the induction of

non‑viable viruses with G‑to‑A‑hypermutated genomes36. owing to integration restraints, both linear and circular forms of unintegrated viral dscDNA accu‑mulate in resting CD4+ T cells. linear, unintegrated DNA is susceptible to integration on cell activation and is considered to be a source of potential viral reser‑voirs14,33 (FIG. 2b). However, its short half‑life — 24 hours to a few days — limits its impact in the generation of a stable pool of infected T cells that carry integrated proviruses37,38. Although non‑integrated viral DNA cannot produce viable particles39, it can generate some RNA transcripts and produce the HIV‑1 negative fac‑tor (Nef) in resting CD4+ T cells40 and macrophages41. This could increase cell activation and therefore facili‑tate HIV‑1 replication, either in the same cell40 or in surrounding cells (through the production of soluble factors by Nef‑expressing macrophages)42.

Stable post‑integration latency is more relevant for HIV‑1 persistence and it has been widely documented in different groups of patients, including naive and treated adults, children and elite controllers43–46. The reservoir of latently infected CD4+ T cells carrying replication‑competent HIV‑1 genomes is established in primary infection47. It has been estimated to comprise 106–107

cells in asymptomatic patients48, whose infected naive CD4+ T cells can harbour an average of 3–4 copies of the integrated provirus per cell49. These cells do not progress to complete viral replication unless they are activated50, and their stability and long half‑lives represent major obstacles to HIV‑1 eradication51,52.

The activation of resting CD4+ T cells harbouring linear, non‑integrated DNA14,33 and the persistence of proviral DNA in memory T cells that return to a quies‑cent state after activation are the two proposed sources of integrated proviral DNA in latently infected cells53,54. However, it is difficult to conceive that cells could sur‑vive the massive viral replication that is induced in activated CD4+ T cells. As an alternative, memory T cells could become infected during the decay phase of activation, therefore allowing viral integration with no further progression to active replication55. Recently, it has been proposed that infected memory T cells can be expanded by homeostatic proliferation driven by interleukin‑7 (Il‑7) or low‑level proliferation driven by antigens56. As only a fraction of latently infected cells are naive T cells57, alternative mechanisms for latent integration must exist. one possible explana‑tion could be the contribution of infected thymocytes and suboptimal activation pathways58.

Viral replication in activated T cells. The phenotypic changes that are induced by immune activation in CD4+ T cells provide an optimal environment for robust HIV‑1 replication59. expression of CCR5 allows viral entry, and efficient retrotranscription takes place owing to high nucleotide pools and APobeC3G inhi‑bition36. Following integration into the host genome, the expression of cellular transcription factors and viral Tat increases transcription and elongation of the provirus, respectively, through the recruitment of PTeFb to the lTR promoter29. CD4+ T cells that are

Box 1 | Lymphocyte subpopulations and HIV‑1 replication

• Naive T cells are a population of T cells that have not yet contacted the foreign antigens that they are committed to identify and therefore are not activated.

• Activated T cells are those that initiate clonal proliferation after their T cell receptors recognize non-self peptides that are presented by the class II major histocompatibility complex. T cells can also be activated by inflammatory mediators, in particular cytokines and chemokines, and through the engagement of Toll-like receptors that recognize pathogen-associated molecular patterns65,177.

• Effector T cells are naive T cells than have been activated.

• Memory T cells are those effector T cells that do not die in the process of the immune response but return to an arrested state, waiting for the same antigen to reappear.

• Resting T cells are naive or memory T cells that are at the G0 state of the cell cycle, that have an extended lifespan and that lack T cell activation markers on the cell surface.

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Macrophage HIV-infectedmacrophage

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Elite controllerA patient infected with HIV whose immune system can limit viral RNA to below 50 copies per ml for at least 12 months in the absence of highly active antiretroviral therapy.

activated by HIV‑1, other foreign antigens, inflamma‑tory mediators or Toll‑like receptors become particu‑larly susceptible to HIV‑1 infection60,61. In this context, a vicious cycle of immune activation and HIV‑1 spreading is established (FIG. 2b), and activated CD4+ T cells that are largely located in the gut-associated lymphoid tissue become severely depleted during the course of the disease62–65.

Low-level viral replication in resting T cells. low‑level HIV‑1 replication in CD4+ T cells that lack activation markers has been described in tissues from macaques and patients infected with the virus66,67. Therefore, unlike peripheral blood CD4+ T cells68, mucosal naive T cells can be productively infected by HIV‑1. Several findings support the role for suboptimal activation in driving the increased susceptibility to HIV‑1 infection and the low‑level replication in resting CD4+ T cells. In the absence of T cell receptor (TCR) engagement, a combination of inflammatory cytokines can trigger HIV‑1 replication69,70,

and a similar role has been proposed for Il‑7 (REFs 56,71). In addition, macrophages expressing Nef or activated through CD40 ligand (CD40l) can produce soluble intra‑cellular adhesion molecules (ICAms) and CD23, which in turn increase the susceptibility of resting CD4+ T cells to infection42. Recently, it has been reported that conditioned medium from ex vivo‑cultured tonsil tissue enhances the infection of naive‑tissue CD4+ T cells by preventing APobeC3G inhibition72. These findings emphasize the potential role of soluble factors and cytokines that are produced in the tissue environment in modifying sur‑rounding lymphocytes and increasing their susceptibility to HIV‑1 infection.

Dendritic cells and macrophages as viral reservoirs. The existence of unrecognized in vivo reservoirs that contrib‑ute to persistent viral replication has renewed the inter‑est in the study of HIV‑1 infection in macrophages and dendritic cells. macrophages are susceptible to HIV‑1 infection and are productively infected in both HAART‑treated and untreated patients73,74. Viral replication in macrophages is characterized by longer persistence of unintegrated DNA41, production of chemokines and soluble mediators75 and generation of infective viral par‑ticles76. Interestingly, in macrophages HIV‑1 assembly can occur both at the plasma membrane and in intracellular compartments, such as late endosomes and multivesicu‑lar bodies. The relevance of these compartments for viral production in vivo is still unknown77–79.

Dendritic cells (DCs) also play a key part in HIV‑1 propagation, through the capture of viruses by the receptor DC‑SIGN (DC‑specific ICAm3‑grabbing non‑integrin) as well as through efficient HIV‑1 transmission to T cells at the virological synapse. In addition, DCs can be infected in vitro and lead to DC‑SIGN‑mediated transinfection of CD4+ T cells. However, the role of infected DCs as poten‑tial reservoirs in vivo remains controversial80. Finally, fol‑licular DCs in lymphoid tissues are specialized in trapping and retaining antigens, including HIV‑1 virions, on their surfaces in the form of immune complexes81. Accordingly, follicular DCs may archive HIV‑1 viruses for months or years and provide another mechanism for viral persist‑ence82. unfortunately, research on DCs and macrophages is beset with technical limitations, and their precise roles as long‑term reservoirs are still unknown.

Molecular mechanisms of HIV‑1 latencySite and orientation of the integrated provirus. most mechanisms to maintain HIV‑1 latency operate at the transcriptional level; for example, the chromosome

Figure 2 | HIV‑1 replication in different cellular environments. a | After stimulation with specific antigens, naive CD4+ T cells begin clonal proliferation to produce effector T cells in an attempt to destroy the pathogen. As the antigen wanes, T cells undergo apoptosis or turn into memory cells that remain quiescent until they contact the same antigen, eliciting a more robust immune response. b | After HIV‑1 infects resting, naive T cells, these cells can carry partially transcribed, unintegrated viral double‑stranded cDNA (dscDNA) that can be degraded in the absence of activation. However, if these cells are stimulated by their cognate antigen they begin clonal proliferation, which allows the integration of viral dscDNA into the host cell genome and active HIV‑1 replication. As the antigen fades, most HIV‑infected effector T cells die by apoptosis, but some remain as memory T cells, carrying a permanent provirus in their genome that undergoes viral replication if the cell is stimulated again with the same antigen. In the case of suboptimal activation, the partially transcribed, unintegrated viral dscDNA can be completed and successfully integrated into the host cell genome, inducing a post‑integration latent state. Moreover, a continuous low‑level replication occurs in these cells even in the absence of activation, as is also the case in highly stable cells such as macrophages.

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Gut‑associated lymphoid tissueThe intestinal mucosa- associated lymphoid tissue that constitutes 70% of the whole immune system and may be the main site of HIV-1 activity, despite the use of highly active antiretrovial therapy.

Transcriptional interferenceInterruption of RNA transcription that is caused by adjacent active promoters owing to the competition for transcription factors or the collision of RNA polymerase II elongation complexes.

environment at the site of integration and the avail‑ability of viral and host factors can have substantial influences on viral latency (BOX 2). Proviral integration can occur at different sites and not entirely at random. Specific sequences at the ends of dscDNA are required to generate an efficient PIC15 that seems to be preferen‑tially integrated into chromosomal regions that are rich in expressed genes83. However, there are strong biases that obstruct the access of the PIC to target DNA, such as the wrapping of host DNA in nucleosomes or the presence of already bound transcription complexes84. Integration into outward‑facing DNA major grooves in chromatin is favoured85, and most inducible HIV‑1 proviruses can be found in intronic regions of the most highly expressed host genes86,87, probably owing to the increased chromatin accessibility of these regions84

(FIG. 3a). However, viral replication from these pro‑viruses can suffer intense transcriptional interference because of the orientation of the proviruses or their proximity to a stronger, host gene promoter88 (FIG. 3b,c). Transcriptional interference of HIV‑1 gene expression may have a role in the establishment of latency89,90 and can occur between active divergent promoters, owing to the lack of factor recruitment to the pre‑initiation complex88, and between active convergent promoters, owing to the collision of two RNAPII complexes during elongation, which can lead to the premature termina‑tion of the transcription of one or both complexes89,90. Convergent transcription could also result in elonga‑tion of both viral DNA strands and the subsequent for‑mation of double‑stranded RNA (dsRNA), which might silence proviral transcription through RNA interfer‑ence (RNAi)91, RNA‑directed DNA methylation92 or the generation of antisense RNA93.

by contrast, when the provirus is in a parallel orien‑tation to the host gene, read‑through transcription may repress viral replication by two mechanisms (FIG. 3c). Firstly, it can block the access of transcription factors or displace already bound proteins87,94. Secondly, ongoing transcription from an upstream host promoter can pre‑vent the assembly of the pre‑initiation complex on the 5′ lTR95: the upstream transcription fails to terminate, and RNAPII reads through into the downstream HIV‑1 promoter, interfering with initiation. HIV‑1 sequences are included in the primary transcript of the host gene

and degraded with the rest of the intron86. Despite the universal inhibitory effect between two conver‑gent or divergent promoters, results from two parallel promoters can vary depending on the system87,88,95,96. Read‑through transcription may also increase gene expression of proviruses, owing to the effect of splicing complexes on adjacent parallel promoters, the removal of repressors upstream of the viral transcription start site or alterations in DNA topology88. Together, these data imply that the orientation‑dependent regulation is highly variable and relies on the rate of 5′ lTR occupancy and on the rate of host gene elongation89,97.

Inducible proviruses can also be integrated outside genes into long intergenic regions or gene deserts, which are expected to be heterochromatic or enriched in binding sites for transcriptional repressors98. In fact, integration into alphoid repeats located in centromeric heterochromatin regions is a rare event in primary cells84,99 and strongly disfavours HIV‑1 gene expres‑sion100, promoting pre‑integration latency101. by con‑trast, short intergenic regions commonly host stably expressed proviruses87.

Epigenetic silencing of HIV-1 transcription. Dynamic modifications of chromatin structure and nucleosome remodelling near the 5′ lTR also influence HIV‑1 repli‑cation. Post‑translational modifications of histone tails modulates chromatin rearrangements, with a high‑order chromatin structure reducing the accessibility of transacting factors and RNAPII. Hypoacetylation of histones by histone deacetylases (HDACs) correlates with transcription repression, whereas hyperacetyla‑tion by histone acetyltransferases (HATs) induces transcription activation102 (FIG. 4a). Accordingly, the nucleosome structure may lead to viral latency in CD4+ T cells with integrated proviruses. Deacetylation of histones may remodel nucleosome 1, resulting in HIV‑1 transcriptional repression. Transcription factors such as yin and yang 1 (YY1)103 and late SV40 factor (lSF; also known as TFCP2)104 repress HIV‑1 replication in infected CD4+ T cells by recruiting HDAC1 to the repressor complex sequence located at position –10 to +27 nucleotides in the lTR. other host transcription factors, such as NF‑κb subunit p50 homodimers105 or the C‑promoter binding factor 1 (CbF1; also known

Box 2 | Mechanisms for the generation of HIV‑1 latency

• Transcriptional interference, owing to the site and orientation of the provirus in the cell chromosome.

• Epigenetic silencing, by post-transcriptional modifications (for example, hypoacetylation or trimethylation) of the histone tails that form the nucleosome core and modulate the chromatin structure.

• The absence of nuclear host transcription activators for HIV-1 expression, for example, nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT) and specificity protein 1 (SP1).

• The presence of cellular transcriptional repressors, such as yin and yang 1 (YY1), late SV40 factor (LSF; also known as TFCP2), C-promoter binding factor 1 (CBF1; also known as RBPJ), APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G), inhibitor of NF-κB α‑subunit (IκBα) and COMMD1 (copper metabolism (Murr1) domain-containing protein 1).

• Inefficient elongation of HIV-1 transcripts, owing to the absence of the viral protein Tat and Tat-associated viral factors.

• Unproductive control of viral RNA splicing, owing to the absence of the viral protein Rev.

• Innate host antiviral processes (for example, short interfering RNAs and microRNAs).

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Ac

Ac


a Location of integrated HIV-1 genomes

b Efficient HIV-1 transcription in activated CD4+ T cells

CTD

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HIV-1 LTRHost promoter

HIV-1 LTRHost promoter

c Transcriptional interference

• Convergent promoters: collision of RNA polymerase complexes

• Divergent promoters: lack of factor recruitment

• Parallel promoters: occlusion by read-through transcription of the host gene

Tat

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as RbPJ)106, can also recruit HDACs to the lTR and inhibit viral transcription similarly to YY1 in several cell lines.by contrast, Tat and several cytokines and HDAC inhibitors, such as trichostatin A, decrease HDAC1 occupancy at the repressor complex sequence, induc‑ing nucleosome 1 rearrangement, which activates tran‑scription at the 5′ lTR95 (FIG. 4b). Tat can recruit factors with HAT activity, such as CReb binding protein (CbP; also known as CRebbP), CbP‑associated fac‑tor (PCAF; also known as KAT2b) and human general control of amino acid synthesis protein 5 (hGCN5; also known as KAT2A), to the 5′ lTR, which induces nucle‑osome hyperacetylation in cell lines and in peripheral blood mononuclear cells107 (FIG. 4b). Accordingly, in the absence of Tat, lTR‑associated nucleosomes are hypoacetylated and viral gene expression is silenced, contributing to viral latency in both u1 and Hl3T1

cell lines108. However, reversion to a permissive chro‑matin arrangement by HDAC inhibitors is not enough to induce transcription. Host factors such as NF‑κb, NF‑AT and SP1 must also be recruited to the lTR, forming a complex that recruits other co‑factors includ‑ing HATs, although SP1 has been reported to interact with HDACs in cell lines and in primary resting CD4+ T cells from patients infected with HIV109. moreover, both NF‑κb and SP1 are also regulated by reversible acetylation110,111.

Although histone acetylation is generally associated with gene activation, histone methylation can be asso‑ciated with both activation and silencing. Accordingly, methylation of H3 at lysine 4 (H3K4) and H3K36 is found in active genes, whereas methylation of H3K9, H3K27 and H4K20 is associated with gene silencing112. In particular, H3K9 trimethylation, mediated by the histone‑lysine N‑methyltransferase suppressor of variega‑tion 3–9 homologue 1 (SuV39H1), has been correlated

Figure 3 | Influence of provirus integration site and orientation into the host cell genome on HIV‑1 replication efficiency. a | Most inducible HIV‑1 proviruses can be found in intronic regions of the most highly expressed host genes, in obverse or reverse orientation. b | Efficient HIV‑1 transcription is achieved by the formation of an elongation complex that binds to the transactivation response element (TAR), a 59‑nucleotide stem‑loop structure formed at the 5′ end of the nascent viral transcript. The viral protein Tat interacts directly with TAR and recruits cellular factors that increase the functional capacity of RNA polymerase II (RNAPII), such as positive transcription elongation factor b (PTEFb), composed of cyclin‑ dependent kinase 9 (CDK9) and cyclin T1. Cyclin T1 binds directly to Tat and allows its interaction with TAR, and CDK9 hyper‑phosphorylates RNAPII at the carboxy‑terminal domain (CTD) to induce a sustained viral mRNA synthesis. Acetylation (Ac) of Tat by cellular acetyl transferases, such as CREB‑binding protein (CBP) and CBP‑associated factor (PCAF), enhances its activity. c | Viral replication can undergo transcriptional interference, which occurs by different means depending on the orientation of the provirus in relation to a strong host promoter and induces viral latency. The viral transcript is shown in orange and the cellular gene transcript is in blue. DSIF, 5,6‑dichloro‑1‑β‑d‑ribofuranosylbenzimidazole sensitivity inducing factors; LTR, long terminal repeat; NELF, negative elongation factor.

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Basal transcription machineryThe complex that regulates the initiation and elongation of transcription by binding to a core promoter that is located ~50 bp upstream of the transcription initiation site and contains the highly conserved TATA box. The complex consists of RNA polymerase II and several transcription factors and co-activators.

with heterochromatin assembly and with mediating HIV‑1 silencing113 by recruiting heterochromatin protein 1 homologue‑γ (HP1γ; also known as CbX3)107. The co‑repressor CTIP2 (chicken ovalbumin upstream promoter‑transcription factor (CouPTF)‑interacting protein 2; also known as bCl‑11b) also recruits HDAC1, HDAC2, SuV39H1 and the HP1 proteins to establish a

heterochromatic environment that leads to HIV‑1 silencing in several cell lines114. Consequently, latently infected Jurkat cells show highly deacetylated and trimethyl‑ated histones115. on activation, trimethylated H3 levels and the occupancy of the lTR by the HP1 proteins and HDACs fall rapidly, inducing HIV‑1 replication from the reservoirs.

Figure 4 | Epigenetic modifications that regulate chromatin accessibility and influence HIV‑1 latency. a | Histone acetyltransferases (HATs) form a multimolecular complex with histone deacetylases (HDACs) that is recruited to the target promoters and modifies nucleosome conformation around the binding site, along with methyl transferases (MTases) such as SUV39H1 (suppressor of variegation 3–9 homologue 1) and DNA methyltransferase 1 (DNMT1). The balance between HDACs and HATs modifies viral transcription, because recruitment of HATs makes chromatin permissive for HIV‑1 replication, whereas recruitment of HDACs and MTases and the consequent release of HATs leads to a higher‑order chromatin structure that causes transcriptional silencing and latency. b | Disruption of nucleosome 1 (nuc1) by HATs, Tat or HDAC inhibitors unlocks chromatin to allow the binding of the pre‑initiation complex, formed by cellular transcription factors such as nuclear factor of activated T cells ( NFAT), nuclear factor‑κB (NF‑κB), specificity protein 1 (SP1) and activator protein 1 (AP1), along with the basal transcription machinery. The distal HIV‑1 enhancer contains consensus sites for lymphoid enhancer‑binding factor 1 (LEF1) and Ets1 (v‑Ets erythroblastosis virus E26 oncogene homologue 1). The initiation of transcription allows the binding of the elongation complex (formed by Tat, positive transcription elongation factor b (PTEFb) and HATs (CREB‑binding protein (CBP) and CBP‑associated factor (PCAF)). Conversely, deacetylation or methylation of histones may remodel nuc1, resulting in HIV‑1 transcription repression. Elongation is also restricted by negative factors DSIF (5,6‑dichloro‑1‑β‑d‑ribofuranosylbenzimidazole sensitivity‑inducing factors) and negative elongation factor (NELF). PTEFb phosphorylates DSIF, NELF and the carboxy‑terminal domain (CTD) of RNA polymerase II (RNAPII), resulting in enhanced RNAPII processivity and transcription of the full HIV‑1 genome. Ac, acetyl group; H3, histone 3; INR, initiation element; LTR, long terminal repeat; TF, transcription factor; TFIID, transcription factor II D complex; TAR, transactivation response element; TBP, TATA binding protein.

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Proviral integration can also be affected by histone modifications. In fact, integration favours regions rich in H3 and H4 acetylation as well as H3K4 methylation and disfavours regions rich in H3K27 trimethylation and CpG island methylation85.

Host cell factors with restrictive activity against HIV-1. HIV‑1 gene expression is strongly dependent on host cell transcription factors. one example of such a transcription factor is inhibitor of NF‑κb α‑subunit (Iκbα), which seques‑ters NF‑κb dimers in the cytoplasm by a non‑covalent interaction. on cellular activation, the Iκbα kinase com‑plex is activated through the protein kinase C (PKC) signalling pathway and phosphorylates Iκbα, resulting in its ubiquitin‑mediated degradation. This allows the now free NF‑κb to enter the nucleus and bind cognate sequences in the proviral 5′ lTR116. Accordingly, over‑expression of Iκbα inhibits HIV‑1 replication and can be responsible for the maintenance of latency in resting T cells117,118. Another example is the transcriptional regula‑tor mYC, which binds SP1 at the lTR , forming a complex that recruits HDAC1 to repress HIV‑1 gene expression, thereby maintaining viral latency (REF. 109). SP1 also inter‑acts with Vpr to increase the transcriptional activity of the cyclin‑dependent kinase inhibitor p21, a key regulator of the cell cycle119. Induction of p21 prevents integration of the viral genome and establishes pre‑integration latency by binding to HIV‑1 integrase120.

The inhibition of Tat may also induce latency. Tat activity is regulated mainly through the acetylation of lys28 and lys50. Acetylation of lys28 by PCAF stimu‑lates Tat association with PTeFb121, whereas acetylation of lys50 by CbP122 promotes the dissociation of Tat from the Tat–TAR–cyclin T1 complex123 and causes it to bind instead to PCAF during early phases of transcript elon‑gation124. This Tat–PCAF complex is then recruited to the elongating RNAPII. Some cellular proteins can affect the acetylation state of Tat, modulating its activity. Tumour protein p73 binds to Tat, preventing its acetyla‑tion at lys28 by PCAF124, whereas sirtuin 1 (SIRT1)125, a class III HDAC, acts as a specific Tat deacetylase, increas‑ing the quantity of Tat that is available to act as a tran‑scriptional activator. In addition, CDK9 acetylation by hGCN5 and PCAF notably reduces the transcriptional activity of PTeFb, promoting HIV‑1 latency126. CDK9 is deacetylated on T cell activation, inducing RNAPII hyperphosphorylation and HIV‑1 replication. other post‑transcriptional modifications of Tat, such as the methylation of residues Arg52 and Arg53 by the arginine N‑methyltransferase 6 (PRmT6)127,128 negatively affect the formation of the Tat–TAR–cyclin T1 complex128.

Finally, specific restriction factors exist to defend the host cell against viral infection. Innate cellular fac‑tors such as CommD1 (copper metabolism (murr1) domain‑containing protein 1)129 and APobeC3G36

impair various early phases of the HIV‑1 life cycle, inducing latency. CommD1 not only interferes with both basal and tumour necrosis factor‑induced NF‑κb activity, blocking HIV‑1 replication in resting lym‑phocytes, but also directly interacts with NF‑κb. Rather than interfering with NF‑κb nuclear translocation, as

Iκbα does130, CommD1 seems to control the duration of NF‑κb recruitment to open chromatin131. APobeC3G strongly inhibits HIV‑1 replication in CD4+ T cells by inducing C‑to‑u conversions in the viral minus strand DNA during reserve transcription132,133. This inhibitory effect is limited to resting cells, in which APobeC3G exists as an active, low‑molecular‑mass ribonucleopro‑tein complex134. T cell activation induces the shift from active APobeC3G to an inactive, high‑molecular‑mass complex that cannot restrict viral infection36. Inactive APobeC3G can be found in tissue‑resident naive T cells, which are permissive to HIV‑1 infection72. This correlates with the fact that HIV‑1 can infect resting CD4+ T cells residing in lymphoid tissues but not those circulating in peripheral blood.

RNAi pathway. The RNAi pathway acts as a barrier against viral replication135 and introduces a new level of complexity to virus–host interplay136 (FIG. 5). First, several cellular microRNAs (miRNAs), namely, miR‑28, miR‑125b, miR‑150, miR‑223 and miR‑382, seem to control HIV‑1 replication by targeting all spliced or unspliced HIV‑1 mRNAs except Nef‑coding mRNA137. These cellular miRNAs are overexpressed in resting CD4+ T cells and inhibit the translation of almost all HIV‑1‑encoded proteins that contribute to viral latency, includ‑ing Tat and Rev138. Second, viral genomes produce viral interference RNAs (viRNAs) that can target viral RNAs (such as LTR139, Nef 140 and Env141), cellular mRNAs and even cellular miRNAs. by targeting its own mRNAs, the virus induces its own latency. Targeting cellular genes such as CD28 (REFs 142–145) leads to modification of the environment in infected cells. However, some of these data remain controversial, as they have been gen‑erated by overexpression of miRNA or viRNA but have not been confirmed in more physiological conditions146. Finally, cellular miRNAs can target factors involved in HIV‑1 replication; for example, miR‑17, miR‑5p and miR‑20 silence PCAF, resulting in the downregulation of HIV‑1 expression. overall, these RNAi mechanisms cause inhibition of viral gene expression or deficient HIV‑1 propagation.

As is the case with other immune defence mechanisms, HIV‑1 has to counteract RNAi pathways to ensure its own replication. Viral products interfere directly with the cellular RNAi machinery through three different mechanisms. The first is the Tat‑mediated partial repression of the ability of Dicer to process precursor dsRNAs into small interfering RNAs (siRNAs)147 through a physical interaction between the Dicer helicase domain and a 30–72‑amino‑acid region of Tat148, leading to the suppression of the RNAi pathway147. However, the upregulation in cellular miRNA expression that is triggered following HIV‑1 infec‑tion seems to contradict the inhibition of Dicer by Tat. Furthermore, the change in miR‑17 levels that is induced by Tat in infected cells has not been confirmed by other authors146. The second mechanism involves the viral TAR sequence, which sequesters the Dicer‑interacting protein TAR RNA‑binding protein 2 (TRbP2), preventing the for‑mation of a functional RNA‑induced silencing complex (RISC)141,149,150. The third mechanism involves the

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http://www.uniprot.org/uniprot/Q8N668

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Multiple splicedviral mRNAs

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modification of some miRNAs151,152. In particular, miRNAs derived from TAR following asymmetrical processing of Dicer can regulate gene expression in vivo153.

In conclusion, HIV‑1 seems to modify the host cell miRNA expression profile, but the ultimate consequences of these changes and the underlying mechanisms are still undefined. both cellular and viral miRNAs could be involved in maintaining HIV‑1 latency or in control‑ling low ongoing viral replication. In addition, HIV‑1 has

developed strategies to overcome the cellular miRNA restriction machinery or enhance the expression of certain favourable miRNAs to achieve full replication154.

Sources of plasma viraemiaIn patients who have not been treated with HAART, viraemia is mainly generated by repeated cycles of infection–replication in activated CD4+ T cells. Activation of latently infected T cells and replication

Figure 5 | The microrNA pathway can modulate both cellular and viral gene expression. DNA from intergenic regions, introns or exons encodes pre‑microRNAs (pri‑miRNAs) that are cleaved in the nucleus by the microprocessor complex (consisting of the RNase III endonuclease Drosha (also known as ribonuclease III) and the co‑factor DGCR8 (DiGeorge syndrome critical region 8 gene)) to produce a precursor hairpin structure of 60–70 nucleotides, termed pre‑miRNA, which encodes a single miRNA sequence. This is exported to the cytoplasm by exportin 5. Cytoplasmic pre‑miRNAs are further cleaved by an RNase III endonuclease, Dicer, in concert with the co‑factors transactivation response element‑RNA‑binding protein (TRBP) and protein kinase R‑activating protein (PACT), which remove the loop sequence and form a 21–23 nulceotide miRNA asymmetric duplex. This mature duplex binds the RNA‑induced silencing complex (RISC), which degrades one strand to produce an active, single‑stranded miRNA that can target mRNAs with sequence homology. Targets are marked for full degradation when the complementarity is complete and for translation inhibition when the complementarity is partial. The miRNA pathway can influence HIV‑1 latency through four mechanisms. Cellular miRNAs can target and degrade viral mRNA (step 1). Viral genomes produce miRNAs, called viRNAs, which can target viral RNAs and cellular mRNAs or miRNAs (step 2). Cellular miRNAs can target cellular factors that are involved in HIV‑1 replication (step 3). Finally, viral products can interfere directly with cellular siRNA machinery by hijacking Dicer (mediated by Tat) and TRBP (mediated by TAR). AGO2, argonaute 2.

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in other cell types contribute to only a low proportion of the viral load (FIG. 6). This scenario is supported by classical studies on viral replication kinetics155 that show a two‑phase decay in viraemia after initi‑ating HAART. The first phase reflects viral produc‑tion by activated CD4+ T cells with short half‑lives (roughly 1 day), which are responsible for more than 90% of plasma viral load. The second phase of viraemia corresponds to viral replication from reservoirs with an estimated half‑life of a few weeks, which produce HIV‑1 at lower rates155. It has been proposed that this second reservoir comprises macrophages, resting T cells, infected DCs or viruses trapped in follicular DCs. A third phase of persistent residual viraemia (1–5 copies per ml) can also be detected in patients undergoing long‑term HAART156,157.

The origin of residual viraemia is a matter of con‑troversy, and three hypotheses have been proposed. In the first, low‑level, ongoing replication is suggested to occur as a consequence of non‑fully suppressive HAART158,159 (FIG. 6). In the second, reactivation from a long‑lived reservoir of latently infected T cells is sug‑gested as the cause. The third hypothesis proposes that virus release from a stable, unrecognized reser‑voir leads to low‑level, persistent production of viral particles without additional cycles of infection but leading to the detection of viral RNA160.

Residual viraemia157,161 has been suggested as a proof of ongoing viral replication cycles, but its control following HAART intensification162,163 has not been confirmed in recent studies164. Current pharmacody‑namic models that assess the antiretroviral inhibitory potential in single‑round infections165 indicate that current HAART regimens can fully control ongo‑ing cycles of HIV‑1 replication. Nevertheless, low pharmacological activity due to reservoirs located in anatomical sites with limited penetration for anti‑retrovirals, such as the gut or central nervous system, cannot be excluded. In fact, HIV‑1 RNA has been found in the lymph nodes166 and the gut167,168 despite undetectable plasma viraemia.

It has been argued that the maintenance of viral DNA in peripheral blood mononuclear cells and gut‑associated lymphoid tissue after an initial decay supports ongoing HIV‑1 replication158,169,170. A good correlation between DNA levels and viral suppres‑sion has been found in different situations162,171–173; therefore, stable DNA could possibly be viewed as a marker of persistent viral replication despite HAART. However, maintained DNA levels could also be a consequence of the stability of the latent reservoir in resting CD4+ T cells51,52, including replication‑incompetent integrated DNA copies48. In fact, the initial decay of total viral DNA levels is mainly due to a decrease in linear non‑integrated DNA, whereas integrated proviral DNA remains stable and repre‑sents the larger DNA reservoir in patients treated with HAART170.

Conflicting results regarding the origin of residual viraemia have been obtained from viral evolution studies and comparisons of plasma viruses and proviral DNA


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Viral replication in non-treated patients with HIV infection

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New cycles ofinfectionInfection of new targets

T celldestruction

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Figure 6 | Sources of viral replication in patients infected with HIV‑1 and strategies to control residual viraemia. (Left panel) Plasma viraemia in non‑treated patients infected with HIV‑1 is mainly generated by repeated cycles of infection–replication in activated CD4+ T cells; robust replication occurs in these cells, and they are highly susceptible to de novo infection. Reactivation of latently infected T cells, persistent replication in other cell types such as macrophages and dendritic cells (DCs) or viruses being trapped in follicular DCs contribute in lower proportions to viraemia. (Right panel) CD4+ T cells carrying latent proviruses can become activated and contribute to residual viraemia in patients treated with highly active antiretroviral therapy (HAART). Reactivation of latent reservoirs (REACT) has been proposed to purge latent viruses and force HIV‑1 eradication. But this reactivation should always be combined with HAART to block the spread of infection by new viral particles that are produced from the activated reservoirs. The use of immunosuppressive drugs (IM SUPP) can contribute to the maintenance of T cells in a resting state, to avoid the expansion of reservoirs through the proliferation that occurs in latently infected T cells. HAART intensification (HAART INT) could achieve complete suppression of the residual viraemia that is caused by ongoing replication in T cells that escape from HAART control. Finally, the use of antibodies coupled to drugs or treatment with immunotoxins (TOXIN) are potential strategies for selective killing of the remaining unidentified cellular reservoirs that produce residual viraemia even in patients treated with fully suppressive HAART regimens. In this case, although intensification of HAART could potentially control long‑lasting viral replication from this reservoir, resistance to cytopathic effects in these persistent cells would be a major obstacle to complete viral eradication.

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in CD4+ T cells. Viral evolution in proviral DNA and cross‑infection among different lymphocytic compartments were detected158,163,168, suggesting that replication occurs in CD4+ T cells. by contrast, characterization of residual viraemia at the clonal level160,174 has shown that although some plasma viral clones share sequences with proviruses that are inte‑grated in resting T cells, most plasma clones (termed predominant plasma clones) do not come from circulat‑ing CD4+ T cells. In addition, predominant plasma clones are homogeneous populations that remain genetically stable for years, suggesting that they are not produced by ongoing cycles of viral replication but originate instead from long‑lasting cells that never enter blood circulation, such as tissue CD4+ T cells, chronically infected tissue macrophages or dividing monocyte–macrophage‑lineage stem cells160. In sum‑mary, although low‑level ongoing replication in CD4+ T cells cannot be fully excluded in all patients treated with HAART, residual viraemia seems to be fuelled by a different reservoir. based on these hypotheses, several strategies have been proposed to tackle viral reservoirs (see below) (FIG. 6).

Therapeutic strategies for eradicationHAART intensification. The aim of the HAART inten‑sification strategy is to achieve a complete suppression of residual viraemia. However, recent intensification studies failed to decrease residual viraemia any more than normal HAART164, suggesting that current regimens can halt ongoing cycles of viral replica‑tion effectively. Nevertheless, the approval of potent drugs targeting CCR5 (REF. 175) and integrase176 has raised new expectations for successfully decreasing the reservoir size in patients with primary infection in particular167,173. This strategy must be analysed in clinical trials before we can conclude whether current HAART combinations have achieved the maximal effect in controlling HIV‑1 replication or whether an intensification of the effect is still possible.

Immunosuppressants. The addition of immunosup‑pressants to HAART combinations has been pro‑posed to decrease the activation of CD4+ T cells and reduce their susceptibility to viral infection and rep‑lication. Corticosteroids177, mycophenolic acid178,179, cyclosporine A180, hydroxyurea181,182 and thalido‑mide183 have been assayed in several clinical trials. These compounds directly decrease the activity of transcription factors such as NF‑κb or NFAT, protect the cell from apoptosis or reduce the production of pro‑inflammatory cytokines such as tumour necrosis factor, thereby reducing immune stimuli, HIV‑1 rep‑lication and cell destruction. Some benefit from their application has been observed in small trials, but drug withdrawal causes viral load to return to basal levels. The most interesting results were found in patients who were recently infected with the virus and treated with cyclosporin A, because CD4+ counts were higher than in patients treated only with HAART (although no changes in viral load were observed)180. However,

the general use of immunosuppressants is not justi‑fied because of their toxicity, although their efficacy in certain situations, for example primary infection, should be studied in controlled clinical trials.

Reactivation of latent reservoirs. The combination of HAART with new therapeutic agents that can reacti‑vate latent reservoirs would lead to HIV‑1 eradication. However, the use of cytokines, antibodies against CD3, non‑tumorigenic phorbol ester derivatives and HDAC has shown limited success. Despite initial optimistic findings184, combined treatment with Il‑2 and HAART has not reduced HIV‑1 reservoirs, and viral rebound has been systematically observed185. A combination of anti‑bodies against CD3 and Il‑2 has proved to be highly toxic, without a clear benefit and is not further advised for HIV‑1 treatment186. Interestingly, Il‑7 can reacti‑vate HIV‑1 in latently infected cells in vitro through the induction of the Janus kinase–signal transducer and activator of transcription (JAK–STAT) signalling path‑way71. In vivo, Il‑7 increases the TCR repertoire and expands both naive187 and memory56 T cells, making this cytokine an attractive candidate for future studies. The use of different chemical compounds targeting the PKC signalling pathway has also been proposed as a means of reactivating viral reservoirs. Prostratin increases HIV‑1 transcription in latently infected T cells through PKC activation and induction of NF‑κb and SP1 (REF.

188). Prostratin also downregulates HIV‑1 recep‑tors, which has the additional advantage of decreas‑ing the risk of re‑infection. This non‑tumorigenic phorbol ester has been advanced in clinical develop‑ment, and recent synthesis made the drug available for clinical trials189. other compounds, including phorbol‑13‑monoesters190, the jatrophane diterpene SJ23b191 and hexamethylbisacetamide192, induce potent reactivation of HIV‑1 in vitro and could be future candidates for clinical development. The use of antibodies coupled to drugs and treatment with immunotoxins are other strategies proposed for the selective killing of infected cells. A combination of immunotoxins and agents that induce viral reactivation has proved to be useful for HIV‑1 eradication in cultures of lymphocytes from patients193 and in animal models194. unfortunately, the failure of the initial phase I trial to reduce viral load and the toxic side effects of this treatment precluded its further development195, but this approach could be envisaged in the context of minimal residual disease to selectively destroy hidden viral reservoirs that escapes from HAART. Finally, HDAC blocking is an attrac‑tive potential means of inducing broad reactivation of HIV‑1 reservoirs. Although promising results in the reduction of HIV‑1 reservoirs were achieved using the HDAC inhibitor valproic acid196, these results have not been replicated197,198.

Perspectives and future directionsDespite its effectiveness, chronic treatment with antiret‑rovirals is not devoid of long‑term toxicity problems. Therefore, the challenge in the field of HIV‑1 treatment is to find a cure, which implies that either the virus must

Predominant plasma cloneThe main plasma clone of infected cells that is responsible for most of the residual viraemia in patients on highly active antiretroviral therapy. This clone is replication competent and shows a specific sequence for each patient that cannot be easily found in the patient’s activated or resting CD4+ T cells.

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be eradicated or treatment must be able to be stopped without notable viral rebound. A better understanding of HIV‑1 latency and persistence can provide clues for the development of new strategies for viral eradication. The study of latency had been hampered by technical limita‑tions, but new approaches have allowed the characteriza‑tion of persistent viruses in patients treated with HAART and the study of infected CD4+ T cells at the single‑ cell level. The finding that residual viraemia does not come from circulating, latently infected CD4+ T cells but from an unknown reservoir represents a major obstacle for eradication strategies, including the use of drugs to reactivate latent viruses. Therefore, the identification of these cells is key for understanding HIV‑1 persistence.

Knowledge of the cellular elements that restrict retroviral replication and actively inhibit the viral transcription machinery and an understanding of

the complex interactions between HIV‑1 and cellular and viral siRNAs provide a new paradigm for HIV‑1 latency. As a result, latency should be considered not merely a passive process but, rather, an active process that is tightly regulated by cellular and viral factors. New insights into the molecular mechanisms of HIV‑1 latency have led to the characterization of targets that will be useful for designing new drugs. In particular, the modification of chromatin conformation through HDAC inhibitors and the activation of kinase path‑ways leading to the activation of transcription factors are both attractive possibilities for specific drug devel‑opment. Finally, cellular factors that are involved in the maintenance of latency, such as APobeC3G and miRNAs, or factors required by the virus to complete PIC transport and integration, such as emerin and leDGF, are also new targets for drug development.

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AcknowledgementsWe thank P. Perez‑Romero, M. J. McConnell, S. Moreno and A. Alcamí for helpful suggestions. The research of our labo‑ratory is supported by grants from the European Union (EUROPRISE), the Instituto de Salud Carlos III (RETIC RD06/0006 and the Intrasalud programme), the Comunidad de Madrid (VIRHOST Network) and FIPSE (Fundación para la investigación y prevención del SIDA en España).

DATABASESUniProtKB: http://www.uniprot.orgAPOBEC3G | CBP | CDK9 | COMMD1 | cyclin T1 | Dicer | HDAC1 | hGCN5 | HMG1 | HP1γ | IκBα | IL‑2 | IL‑7 | LEDGF | LSF | Nef | PCAF | Rev | SP1 | Tat | Vpr | YY1

FURTHER INFORMATIONJosé Alcamí’s homepage: http://www.isciii.es/htdocs/centros/microbiologia/unidades/micro_unidad_inmunopatologiasida.jsp

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VH1 latente proporciona pistas para la erradicación de depósitos a largo plazo

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