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Antiretroviral therapy has managed to reduce HIV viral levels to that of below detection. However it is a far cry from the eradication of the virus from the human body. This is a difficult task to achieve as reservoirs of infection; which exist due to HIV latency, are difficult to purge completely. This essay discusses the origins and classification of this virus, how it infects the cells of the immune system, the concept of viral latency and establishment of reservoirs, the various mechanisms of latency and how reservoirs could be potentially purged.
Introduction to Human Immunodeficiency Virus
HIV/AIDS (Acquired Immunodeficiency syndrome) has emerged as one of the most devastating diseases of modern times affecting over 60 million individuals and claiming over 25 million lives worldwide. HIV was first discovered in the USA in the late 1980s and it was revealed that it stemmed from a simian immunodeficiency virus (SIV), found in sub-Saharan African primates that had crossed over into human population. The two types of HIV present; HIV-1 and HIV-2 stem from different primate reservoirs: the former being the chimpanzee (Pan troglodytes) and the latter being the sooty mangabey (Cercocebus atys). The groups of HIV-1(accounting for the bulk of the cases worldwide) are M (main), N (non-M, non-O) and O (outlier) and are distantly related. The sub-types describe lineages within the M group and are listed A to D, F to H, J and K and sub-subtypes characterize lineages that are distant, but not enough to warrant christening of a new subtype( F1 and F2). Recombination between subtypes of M group has resulted in the addition of circulating recombinant forms (CRFs) to the nomenclature (examples include CRF01 and CRF02). HIV-2 is found almost endemically in Africa and its spread is limited due to lowered viral loads as compared to HIV-1.
How HIV undermines the human immune system
HIV-1's life cycle is described by its initial entry and integration into the cell followed by viral replication and budding of virions. The HIV-1's gp120 protein exploits CD4 and chemokine receptors CXCR4 and CCR5. In fact, deletion of 35 bases in the gene encoding CCR5 gives a receptor that doesn't reach the cell surface, thereby granting resistance to infection. Once the virion enters the cell, it must be uncoated. This is accomplished by phosphorylation of matrix protein by MAP kinases and by the viral proteins nef and vif .The contents of the viral core which include the RNA, reverse transcriptase, tRNA primers and integrase passes into the cytosol of the cell where reverse transcription of the RNA gives rise to a linear double stranded DNA(dscDNA). This reverse transcriptase complex now starts forming the pre-integration complex (PIC) which comprises the synthesized dscDNA, matrix, integrase, the reverse transcriptase, the viral protein r (Vpr), High mobility group protein B1 and the lens epithelium-derived growth factor. The Vpr is implicated in mediating virus import into the pore using the cell's cytoskeletal structures like microtubules and motor protein dyenin. Once inside, the PIC can integrate with the chromosome which is mediated by integrase or alternatively form one or two LTR circles.
The LTR containing provirus behaves akin to a gene, with the 5' LTR playing the role of a eukaryotic promoter and the 3' LTR serving as a site of termination. Transcription factors like nuclear factor- ?B (NF-?B), nuclear factor of activated T cells (NFAT) and E26 transformation-specific transcription factor family are employed to improve the binding of RNA polymerase II (RNAPII) to the TATA box to begin transcription. The transcript produces a transactivation response element (TAR) that then binds to viral transactivator (Tat). This interaction calls cellular cyclin dependant kinase 9 (Cdk9) to the site and longer transcripts are produced. HIV-1 manipulates the local cellular environment via the nef gene which manages to inhibit apoptosis, lower CD4 T-cell responses by exploiting Fas-FasL mechanisms and inhibit p53. The polypeptides thus formed mature into HIV virions and bud off the infected cell and the infective process repeats.
HIV-1 and Latency
HIV-1 is very difficult to cure for many reasons. One reason is attributed to the high error rate in the reverse transcriptase due to which many mutants are cropping up. Another reason is latency, whereby reservoirs of infection are created in the body which leads to reinfection after treatment is discontinued. This latency makes for HIV-1 eradication a herculean effort and therefore requires a thorough introspection into its mechanisms.
When a patient is started off on anti-retroviral treatment, the number of copies per ml of the HIV-1 reduces until it is passes under the lower limit of detection (50 copies/ml). This occurs in four phases. The first phase comprises of a population of CD4+ T-cells with a half life of two days. The second phase encompasses other infected cell types including macrophages and dendritic cells (DCs) with a half-life of upto 4 weeks. Phase three has infected cells with a half life of many weeks and phase four has no steady noticeable decline due to reactivation of latently infected memory T-cells. This shows that reservoirs or sites of prolonged viral persistence other than the primarily infected sites causes latency and hurdles to HIV-1 eradication by medication.
Sources of latency
Resting lymphocytes (the long living T-cells that do not express activation markers on the cell surface) are implicated as sites that encourage latency. These cells do not express CCR5 and therefore HIV-1 targeting is impacted and unconducive factors like low ATP and nucleotide levels negatively impacts reverse transcription and PIC importing events. Also the virus must bypass cytoplasmic apolipoprotein B mRNA -editing enzyme catalytic polypeptide- like 3G (APOBEC3G), which interferes in reverse transcription via hypermutation. All this leads to unintegrated dscDNA. This constitutes pre-integration latency. However; this linear dscDNA cannot be a reservoir of infection owing to its half life is a few days atmost. Other organs like the spleen and tissues like the gut associated lymphoid tissue (GALT) are candidates. Also it has been discovered that DCs, macrophages, the renal epithelium and the central nervous system (CNS) are reservoirs of latently infected cells.
The perennial source of post-integration latency of HIV-1 is infected naïve CD4 T-cells and infection occurs once they are activated. Also the infected memory T-cells (the T-cells that are not apoptosed but serve to mount a response to the antigen if encountered again), owing to their long live span constitutes a stable reservoir for latency. Thus latency serves to reinfect the host and therefore its mechanisms be analyzed and steps to counter it should be planned.
Molecular mechanisms in latency of HIV-1
It was found that HIV-1 integrates with actively transcribed genes, heterochromatin and chromosomes with little gene content. If HIV-1 integrates in the same polarity of an upstream promoter that is transcribing a host cell gene, it can negatively affect the activity of a promoter found downstream by disrupting the binding of transcription factors to the downstream promoter. However if the reverse scenario were to occur, where the integration of the provirus is reversed with respect to the upstream promoter, interference of transcription occurs due to conflict of polymerases of HIV-1 and that of the host cell. In the scenario with parallel orientation of the pro-virus to the host gene, the transcription of the host gene upstream can interfere in the formation of PIC and the RNAPII might interfere with the 5' LTR. But the process of read through transcription has been implicated in assisting provirus expression. Thus we can see how the positioning of the promoters of the host cell and the provirus affects transcriptional behavior.
Epigenetics (heritable changes in gene expression without DNA sequence modification) plays a role in HIV-1 latency. When the provirus is integrated into heterochromatin, two nucleosomes hide the site that recruits the transcription factors. Post translational histone modification involves addition and removal of acetyl groups mediated by histone acetylltransferases (HATs) (HAT) and by histone deactelyases (HDACs) respectively. The addition of acetyl groups exposes the site and thereby transcription of the integrated provirus occurs. Similarly, the removal of acetyl groups represses transcription. A variety of factors like yin and yang 1and C-promoter binding factor 1 (CBF1) recruit HDACs to the provirus promoter site and prevent transcription. In response the Tat protein of the virus can employ other factors that can induce hyperacetylation at the promoter.
DNA methylation of cytosine-guanine rich regions (CpG) by themselves or in complexes with HDACs is responsible for preventing transcription. The methylation of histone H3 at several lysine residues results in activation or silencing the gene. Trimethylation at lysine 9 in presence of histone-lysine N-methyltransferase suppressor of variegation 3-9 homologue 1 (SuV39H1) has shown silencing of HIV-1.
As seen in the mechanism of HIV-1 infection in the earlier part of the text, transcription factors derived from the host cell such as NF-?B help in transcribing the integrated provirus. The a-subunit of NF-?B responsible for its assembly has an inhibitor named I?Ba which regulates NF-?B's activity by binding to it non-covalently and preventing its translocation. Over expression of I?Ba disallows NF-?B access to the 5' LTR of the provirus and can contribute to latency. Other examples include inhibition of Tat protein by acetylation of lysine residues, methylation of Tat's arginine residues, CDCK9 acetylation and the involvement of APOBEC3G.
MicroRNA (miRNA); processed from pre-miRNA, is a 21-24 nucleotide single stranded non-coding RNA molecule that is implicated in down regulation of gene expression or RNA interference (RNAi). miRNA finds regions of complementarity on the messenger RNA (mRNA) and through a ribonucleoprotein complex, degrades the mRNA, thereby dampening gene expression. An Endoribonuclease Dicer which comprises a helicase and an RNase motif chops up dsRNA to produce small interfering RNA (siRNA) and by association with a RNA-induced silencing complex (RISC), the complementary regions on the mRNA is targeted and cleaved. A point to note is that siRNA is derived from a long dsRNA in contrast to the miRNA which is derived from a hairpin shaped RNA. In addition to RNAi by miRNA /siRNA, HIV-1 inhibits it viral protein production by viral interference RNAs (viRNAs) and induces its own latency. But HIV-1 has methods to stop the RNAi by cellular miRNAs (like miR-17 and miR-20). One way is by Tat interacting with the Dicer helicase and preventing RNAi. Another way is by sequestering the protein that forms the RISC complex and finally by actually modifying the miRNA. The RNAi events interfere in translation of viral proteins and ultimately lead to latency. Further studies will help measure the effect on RNAi on latency.
Studying HIV-1 latency
When T-cells encounter the antigen, a fraction of them return to a resting phase and become memory T-cells as mentioned previously. Post integrative latency is established in these memory T-cells and as seen the events that contribute to the latency are many in number and complex in their own right. There are quite a few difficulties in studying latency in vivo. The hurdles include limited number of latently infected cells, difficulty in cell enrichment and dysfunctional post integrated provirus and effective cell culture models to study latency in vitro. Using cell cultures like the ACH2 T-cell line, promonocytic and J-Lat cell lines HIV-1 transcriptional activity has been studied. Several scientists have also devised models that ensure greater numbers and survival of latently infected cells. These models also study the reactivation of the virus from its latent state. For example the memory T-cells kept alive by protein Bcl-2 were reactivated by a HIV-1 vector and thus the mechanisms leading to infection from the latent state could be examined.
Dealing with latency
A number of chemical compounds like Protein kinase C (PKC) agonists, phorbol esters prostratin and 12-deoxyphorbol 13-phenylacetate (DPP) have reinitiated transcription of HIV-1 in latent CD4 T-cells. The PKC agonists impact the expression of CCR5, required for HIV-1 infection and thereby forces expression of latent proviruses and reduce the incidence of new infection.
Phorbol ester prostratin is another compound that activates PKC, prevents fresh synthesis of provirus while activating latently infected T-cells in lymphoid and myeloid cell lines simultaneously. It is derived from a plant named Homolanthus nutans and is non toxic. The culmination of these advantages makes it an ideal drug to purge the HIV-1 reservoirs.
Immune activation therapy (IAT) mediated by cytokines seeks to encourage expression of latently infected CD4 T-cells and in other reservoirs like macrophages and monocytes. A regimen of Interleukin-6 (IL-6), IL-2 and Tumor necrosis factor (TNF-a) was used to employ IAT in patients undergoing highly active antiretroviral therapy (HAART). Additionally, INF-? was administered along with IL-2 to induce IAT in infected macrophages. But studies indicated that only a partial purge was achieved because recurrence of viremia ensued after the cessation of treatment. Recent studies of IAT with IL-7 have shown that IL-7 helps in preservation of the reservoirs of memory T-cells and has called to attention the validity of IAT in patients undergoing HAART.
The use of HDAC inhibitor (HDACI) like Trichostatin A and sodium butyrate inhibits deacetylation of nucleosomes and encourages transcription of HIV-1 provirus, thereby contributing to purging of reservoirs. The inhibitors are classified by structure into five families and each family and member of the family establishes different selectivity and efficiency and so the class and member of HDACI used for purging HIV-1 reservoirs must be selected carefully to maximize transcriptional activity of HIV-1 while not negatively affecting other HDAC families. HDACIs have been administered in humans for treatment of other diseases like epilepsy (phenylbutyrate) and T-cell lymphomas (Vorinostat) and in case of HIV-1 it has shown down regulation of CXCR4 coreceptors required for HIV-1 infection. But there are causes for concern regarding use of HDACIs as acetylation has been implicated in other cellular functions like DNA recognition and proteinaceous interactions and HDACI can alter these crucial functions. So the use of HDACIs must be done after much deliberation. Similarly the search for histone demethylase inhibitors (HDMTIs) is also ongoing. Hexamethylene bisacetamide (HMBA) has been employed in human clinical trials and has been shown that it causes Cdk9 to gather at the HIV LTR promoter site.
The lives of people infected with the HIV-1 virus have been made manageable with HAART treatment which brings viral levels to less than fifty copies/ml. But it is not sufficient to completely eradicate the virus. The reservoirs of latently infected cells, which include memory T-cells, macrophages, monocytes, the gut associated lymphoid tissue and the central nervous system are established during the initial stages of infection. These reservoirs need to be purged in order for the eradication of HIV-1 from the human body and a number of treatment options from HDACIs to intensified HAART treatments have been proposed and are undergoing testing in clinical trials. The studies on HIV-1 latent cells and its reactivation have shown that it is a complex cellular and epigenetic process with intricate mechanisms that have only recently been uncovered in detail. The best hope humankind has for eradicating HIV-1 is by employing a HAART program coupled with new compounds that purge the reservoirs.