oa Editorial [Hot Topic: Mechanisms of HIV-1 Latency Post HAART Treatment Area (Guest Editors: Lena Al-Harthi and Fatah Kashanchi)]

In the current “Hot Topic” series published in the Current HIV Research, the readers will find a number of intriguing reviews that are related to the current state of HIV latency. These reviews are written by some of the leading investigators who have contributed significantly to the HIV latency field. At the outset, we also would like to acknowledge that many other critical topics related to latency were left out mainly due to space and time limitations. The five reviews start with a manuscript from Mbonye and Karn who describe some of the current complexities related to HIV reservoirs in patients including the estimated long half-life of 45 months. These authors do an excellent job of describing the latest mechanisms that regulate both viral promoter and epigenetic associated phenotypes. They also accurately describe a set of evidence from the literature regarding modes of activation in both cell line as well as primary cells. Finally, some of the other possible latency mechanisms including RNA export are included and later discussed in more detail from a review by Ajamian and Mouland. The excellent figures and diagrams in this particular review also do a great job of describing the state of the art in a snap shot. The second review by Planelles, Wolschendorf and Kutsch describes assays that are required for high throughput screening and how some of these assays could effectively be used to screen for activators of latent virus. The authors discuss issues related to integration profiles and contrast patient samples to cell lines in vitro. Previous work from Planelles' lab described use of labeled virus and its utility in creating primary latent T-cells in vitro. The review also provides a table that gives an overview of various modes of activation using antibodies, chemicals and cytokines. These authors also add new technological advances in the high throughput screening assays both in 96 well as well as 384 well formats. Methods of screening that are the most sensitive for high throughput assays are described and detailed (i.e., fluorescence flow cytometry). These authors further consider latent or persistent HIV reservoirs to likely reside in multiple cell types in vivo therefore complicating the mode of action when trying to activate the latent viruses. The next review is by Tyagi and Romerio that defines multiple modes of latency that could occur at transcription initiation, elongation and some of the post transcriptional regulations. The authors also have a table comparing various in vitro models of latency and viral strains that have been used to create these infections. They cite some of the early work from Gallo's group that detected HIV RNA after various early HAART treatments. They pay close attention to both X4 and R5 viruses throughout the review and the distinction between choosing replication competent and single round virus infection are well described. From some of the literature reviews as well as their own work they conclude the expansion of immunological memory cells may be a slow and step-wise process rather than an “all or none” process. Finally, the authors acknowledge that the literature has focused heavily on a narrow window of latency, mainly on transcription of HIV and other restriction points, including downstream events (post transcriptional) which could become significant targets for controlling latency. The next review is by Ajamian and Mouland which discusses some of the post transcriptional events that may control latency. The authors site a number of important papers including one that identified five cellular miRNA that are up regulated in resting T-cells during latency and control HIV production. The miRNA arena which has a natural segway to RNA helicases are described and could potentially control infection. For instance, the RIG-I pathway which controls the innate immunity of the cell is highly regulated by DExD/H RNA helicases. These RNA helicases also directly bind to HIV proteins including Gag and Rev open reading frames. They control multiple mechanisms including RNA surveillance, cytoplasmic export, translation and stability of genomic RNA. Perhaps one of the best cited examples of RNA helicases and Rev contributing to latency comes from studies done in Astrocytes. All in all, there is considerable evidence for the involvement of RNA helicases that may control latency in cells tested to date. One caveat however, is that very few of these studies utilize primary cells or field isolates of HIV. The final review by Van Duyne et al. describes some of the latest findings in humanized mouse models that could potentially contribute to latency. The review discusses various animal models as well as some of the cell line data that have been utilized regarding latency and further describes newer models using different mouse strains. The discussions on in vivo animal models of latency touch on various tissues that could potentially be source of latent populations. Some of the highlighted areas include lymphoid tissues, CNS, Gut, and reproductive tract. Finally, the future direction section provides a more detailed description of newer animal models and the various cell types including T-cells and macrophages that could be the source of viral reservoirs. Although these reviews describe the current state of the art, many possibly significant mechanisms of latency have still not been fully exploited. For instance, issues related to DNA methylation of HIV DNA [1], modifications of Tat protein including methylation [2-3], presence of virus specific miRNA which could control both transcriptional silencing and mRNA regulation [4-8], a clear distinction between wild type and possible mutant pools from in vivo models, and other more pressing distinctions between definition of latency of T-cells in blood stream (∼2-3% of all T-cells) versus T-cells in tissues such as lymph nodes (∼65% of all T-cells), and the incomplete definition of mechanisms that regulate macrophage latency/persistence in blood stream versus tissues all require further research. Finally, in future more intense research is needed to better describe additional HIV cellular reservoirs and sanctuary sites that exist in vivo other than resting CD4+ T cells and monocytes/macrophages. This is significant in light of the fact that resting CD4+ T cells, monocytes, or other “blood” cell type may not be the source of resurgent HIV, highlighting the presence of additional reservoirs for HIV [9-10]. One such reservoir which remains elusive could be the central nervous system (CNS). HIV invades the brain within weeks of infection and persists in the CNS at a steady state, despite cART in cellular reservoirs such as perivascular macrophages, microglia, and astrocytes [11-13]. Because cART typically has lower penetration effectiveness into the CNS [14], the CNS microenvironment may be a breeding ground for drug resistant HIV and/or low level of HIV replication that can drive persistent inflammation in the CNS. Indeed, despite cART, 52% of HIV infected individuals are diagnosed with a spectrum of neurologic disease that range from mild to moderate neurocognitive disorder to more severe encephalitis/dementia [15]. Along these lines knowledge about HIV latency and cell types in CNS is in its infancy. For instance, CD16+ monocytes and other lymphocytes, including CD4+ T cells infiltrate the brain and disseminate HIV into the CNS [16]. Within the brain, microglia, which are resident macrophages, are the primary target for productive HIV infection. However, considerable evidence from in vitro and in vivo studies also indicate that astrocytes harbor HIV DNA [17-20]. In fact, 1-3% of astrocytes from patients without a diagnosis of HIV-associated encephalitis harbor HIV provirus [17]. The size of this pool is impressive considering that the size of the resting CD4+ T cell pool harboring HIV provirus is quite small constituting approximately 1 cell/million resting CD4+ T cells [21]. Therefore astrocytes may constitute a considerable reservoir for HIV in vivo. Further research is needed on this possible source of latent cells which will be important for any therapeutic or “shock and kill” approach to succeed. REFERENCES [1] Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog 2009; 5(6): e1000495. [2] Sakane N, Kwon HS, Pagans S, et al. Activation of HIV transcription by the viral Tat protein requires a demethylation step mediated by lysinespecific demethylase 1 (LSD1/KDM1). PLoS Pathog 2011; 7(8): e1002184. [3] Van Duyne R, Easley R, Wu W, et al. Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR. Retrovirology 2008; 5: 40. [4] Klase Z, Kale P, Winograd R, et al. HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BMC Mol Biol 2007; 8: 63. [5] Klase Z, Winograd R, Davis J, et al. HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression. Retrovirology 2009; 6: 18. [6] Ouellet DL, Plante I, Landry P, et al. Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element. Nucleic Acids Res 2008; 36(7): 2353-65. [7] Schopman NC, Willemsen M, Liu YP, et al. Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs. Nucleic Acids Res 2011. [8] Weinberg MS, Villeneuve LM, Ehsani A, et al. The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 2006; 12(2): 256-62. [9] Brennan TP, Woods JO, Sedaghat AR, et al. Analysis of human immunodeficiency virus type 1 viremia and provirus in resting CD4+ T cells reveals a novel source of residual viremia in patients on antiretroviral therapy. J Virol 2009; 83(17): 8470-81. [10] Chun TW, Davey RT, Jr., Ostrowski M, et al. Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. Nat Med 2000; 6(7): 757-61. [11] Roberts ES, Burudi EM, Flynn C, et al. Acute SIV infection of the brain leads to upregulation of IL6 and interferon-regulated genes: expression patterns throughout disease progression and impact on neuroAIDS. J Neuroimmunol 2004; 157(1-2): 81-92. [12] Chiodi F, Sonnerborg A, Albert J, et al. Human immunodeficiency virus infection of the brain. I. Virus isolation and detection of HIV specific antibodies in the cerebrospinal fluid of patients with varying clinical conditions. J Neurol Sci 1988; 85(3): 245-57. [13] Clements JE, Babas T, Mankowski JL, et al. The central nervous system as a reservoir for simian immunodeficiency virus (SIV): steady-state levels of SIV DNA in brain from acute through asymptomatic infection. J Infect Dis 2002; 186(7): 905-13. [14] Letendre S, Marquie-Beck J, Capparelli E, et al. Validation of the CNS Penetration-Effectiveness rank for quantifying antiretroviral penetration into the central nervous system. Arch Neurol 2008; 65(1): 65-70. [15] Heaton RK, Clifford DB, Franklin DR, Jr., et al. HIV-associated neurocognitive disorders persist in the era of potent antiretroviral therapy: CHARTER Study. Neurology 75(23): 2087-96. [16] Fischer-Smith T, Bell C, Croul S, Lewis M, Rappaport J. Monocyte/macrophage trafficking in acquired immunodeficiency syndrome encephalitis: lessons from human and nonhuman primate studies. J Neurovirol 2008; 14(4): 318-26. [17] Churchill MJ, Wesselingh SL, Cowley D, et al. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia. Ann Neurol 2009; 66(2): 253-8. [18] Dewhurst S, Sakai K, Bresser J, et al. Persistent productive infection of human glial cells by human immunodeficiency virus (HIV) and by infectious molecular clones of HIV. J Virol 1987; 61(12): 3774-82. [19] Dewhurst S, Bresser J, Stevenson M, et al. Susceptibility of human glial cells to infection with human immunodeficiency virus (HIV). FEBS Lett 1987; 213(1): 138-43. [20] Trillo-Pazos G, Diamanturos A, Rislove L, et al. Detection of HIV-1 DNA in microglia/macrophages, astrocytes and neurons isolated from brain tissue with HIV-1 encephalitis by laser capture microdissection. Brain Pathol 2003; 13(2): 144-54. [21] Siliciano JD, Kajdas J, Finzi D, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 2003; 9(6): 727-8.