Facoltà di Scienze Matematiche, Fisiche e Naturali
Corso di Laurea Magistrale in Biotecnologie Molecolari e Industriali
Excision of human immunodeficiency virus as a novel approach to eradicate the
infection: analysis of intracellular persistence and residual activity of the
cleaved provirus within the cell.
Candidata Relatore
Giulia Matteoli Prof. Mauro Pistello
Prof. J.L. Heeney
This work arises from the cooperation between the Retrovirus Centre (University of Pisa) and the Laboratory of Viral Zoonotics (LVZ) (University of Cambridge).
Summary
1. Abstract ... 5
2. Introduction ... 7
2.1 HIV genome ... 7
2. 2 Epidemiology ... 8
2.3 HIV life cycle ... 9
2.4 Infection ... 14
2.5 Reservoirs ... 22
2.6 Therapies ... 31
3. Aim of this work ... 49
4. Material and Methods ... 50
4.1 Molecular cloning ... 50
4.2 Inserts purification ... 53
4.3 Ligation ... 55
4.4 Transformation of E. coli competent cells ... 55
4.5 Colony Screening ... 56
4.6 sgRNA sequence molecular cloning in pspCas9 plasmid. ... 61
4.7 Sanger sequencing ... 63
4.8 Human Embryonic Kidney 293 (HEK293) cell line ... 64
4.9 VSV-‐G pseudotyped pNL:EGFP/CMV/WPREdU3 viral particles transduction. ... 67
4.10 Viral Titration: Flow cytometry. ... 67
4.11 VSV-‐G pseudotyped pNL4.3:LUC:SCS101-‐Ori and VSV-‐G pseudotyped pNL4.3:Luc:KanR viral particles Transduction. ... 69
4.12 CRISPR/Cas9 application on pNL4.3 co-‐transduced HEK293 cells ... 72
4.13 Concatamer purification ... 72
4.14 Electroporation ... 74
4.16 Concatamer Production ... 76
4.17 Concatamer digestion assay ... 77
4.18 Concatamer transcriptional and translational activity ... 77
5. RESULTS ... 81
5.1 pNL4.3:Luc molecular cloning ... 81
5.2 pspCas9 molecular cloning ... 84
5.3 pNL4.3:Luc:KanR and pNL4.3:Luc:SCS101-‐Ori are infectious on HEK293 cells ... 84
5.4 Characterisation of the concatamer ... 86
5.5 RNA expression of the concatamer ... 90
5.6 Protein Expression of the concatamer ... 90
6. Discussion ... 92
1. Abstract
The Human Immunodeficiency Virus (HIV) is a retrovirus evolved from the zoonotic infections with Simian Immunodeficiency Virus (SIV) that has been discovered in the 80s as the etiologic agent of the AIDS. HIV is the cause of the acquired immune deficiency syndrome (AIDS), which impairs the normal function of the immune system. Nowadays the only therapy available is the antiretroviral therapy (cART) that consists in a cocktail of drugs that interferes with different steps during the HIV replication cycle. The cART therapy has improved the life quality of patients, leading to low viral loads along with a normal CD4+ T-cell count, but cART cannot eradicate the infection. This is actually the main problem: latent infection develops when the virus became transcriptionally inactive, but a low viral production is still maintained, and some cells become viral reservoirs. Latent infection can shift to productive one and, if the cART is interrupted, the infection can spread again in a short time.
One strategy that can be used to remove the HIV from the host consists in the proviral genome eradication via gene editing techniques. Different gene editing (GE) techniques have been developed in the last years including the clustered regularly interspaced palindromic repeats (CRISPRs). The elimination of the proviral DNA from infected cells can be achieved targeting the proviral genome. The full-length proviral sequence can be excised targeting 5’ and 3’ long terminal repeats (LTRs). The CRISPR/Cas9 system cleaves efficiently these regions.
This work is focused on the elimination of the full proviral sequence and on which is the fate of the excised proviral sequence after the targeting of LTRs via CRISPR/Cas9 technique. We have observed that the excised provirus can be re-ligated together in tandem by the host reparation machinery creating circular structures named concatamers that we show persist for weeks after treatment.
The next step, and the main aim of this thesis, is to understand if these structures, that have a rearranged sequence of the LTRs, can have some level of transcription and protein expression.
To study this process we have isolated HIV concatamers in a stable way: we have modified the pNL4.3Luc HIV genome by cloning in its Env sequence two different transgenes: a
bacterial Origin of Replication and an antibiotic bacterial selection. A concatamer carrying these two sequences can act like a plasmid, a genetic structure easy in storing and handling. The concatamers have been transfected in uninfected cells to measure expression activity and establish their persistence.
To understand concatamer activity we evaluated Pol and Gag RNA presence by RT PCR and p24 by ELISA immunoassay, transfecting 293T with concatamers. Our data show a residual transcription of concatamer.
These data shed new lights on the fate of HIV concatamers and cast a warning sign on the whole approach. Should these data been confirmed, excision of provirus to cure HIV infection warrants further studies.
2. Introduction
The Human Immunodeficiency Virus (HIV) is a lentivirus, a subgroup of retrovirus, which causes a progressive infection that, without treatment, evolves in the Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease of the immune system, characterised by the impairment of its functionality and increased susceptibility to opportunistic infections and tumours. HIV is transmitted through contact of infected body fluids, containing free viral particles, with mucosal tissue, blood or broken skin. HIV has been pandemic from 30 years and, since its discovery, a great number of studies have provided us information about its replication cycle and about drugs that are nowadays used in combination to reduce, or suppress, the viral load in infected patients.
2.1 HIV genome
HIV is a lentivirus, a viral group member of the Retrovirus family. As Retroviruses it has the ability to reverse transcribe its RNA based genome into DNA and integrate it into the host genome, modifying it in a permanent way, moreover Lentiviruses are distinguished by the ability to infect also non-dividing cells. HIV genome is ssRNA in positive sense and each virion contains two copies of capped and polyadenylated single-stranded RNA (ssRNA) genome as a dimeric functional unit.
HIVs viruses are divided in two main groups: HIV-1 and HIV-2. HIV-1 is the most pathogenic and it is the one considered here. HIV-1 RNA structure achieves several functions: it activates transcription, initiate reverse transcription, facilitate genomic dimerization, direct HIV packaging, manipulate reading frames, regulate RNA nuclear export, signal polyadenylation, and interact with viral and host proteins (Watts J.M., 2009).
HIV-1 genome, represented in figure 1, is primarily a coding RNA and contains nine open reading frames (ORFs) that produce precursor proteins successively cleaved in 15 functional proteins. From the 5’ to 3’ ends there are the 5’ long terminal repeat (5’LTR), gag (group-specific antigen), pol (polymerase), env (envelope glycoprotein) genes and 3’ LTR.
Figure 1 HIV-1 genome organization. Protein coding regions are shown as grey boxes; polyprotein-domain junctions are depicted as solid vertical lines (Watts, 2009).
Gag encodes for the precursors Gag polyprotein that is processed during viral maturation in capsid protein (CA), known as p24 that composes the viral capsid; matrix protein (MA), spacer peptide 1 (SP1); spacer peptide 2 (SP2), nucleocapsid protein (NC) and P6 protein. Pol gene expresses the reverse transcriptase (RT), RNase, integrase (IN) and HIV protease (PR). Env codes for the envelope glycoproteins, it expresses gp160 protein that is cleaved in gp120 and gp41. Gp120 interacts with CD4 receptors on target cells.
2. 2 Epidemiology
HIV virus probably evolved from a simian immunodeficiency virus (SIV) in the late 19th
century and it’s been pandemic from 30 years.
HIVs are divided in two main viral groups: HIV-1 and HIV-2. HIV-1 is more pathogenic and contributes mostly to the pandemic. HIV-1 develops a high mutation rate due to the error prone function of the reverse transcriptase, in this way it can escape to the host immune system response. HIV-2 is more similar to SIV from which evolves and less pathogenic and confined to west Africa (Maartens G., 2015). There is a difference in treatment response: currently antiretroviral therapy is designed on HIV-1 and some classes of drugs might not be effective on HIV-2. It is thought that the reduced HIV-2 pathogenicity depends on lower levels of viral replication and not to a complete adaptation of SIV to the human host.
From the beginning of pandemic 75 million people have been totally infected by the virus, at present 37 million people are living with HIV, of those 15 million are receiving therapeutic treatment. There are 2 million new infections (90% in adults and 10% in children) and 1.2 million of deaths every year, mostly in the Sub-Saharan Africa.
Thanks to the introduction of the antiretroviral therapy and the prevention this data are considerably improved in the last years. Since 2000 new HIV infection have decreased of 35%, 58% among children, and AIDS-related deaths have fallen by 42% since the peak in 2004 (UNAIDS, 2015).
2.3 HIV life cycle
The HIV-1 replication cycle can be divided into an early and a late phase, it is represented in figure 2. The early phase encompasses the events from the recognition of the cellular target to the viral integration into the host genome. The late phase starts with viral gene expression to the packaging and the release of the viral particle from the infected cells (Freed E.O., 2015).
Figure 2 HIV-1 Replication Cycle and the sites of action of different antiretroviral drugs(Marteens, 2015).
The first step in the early phase is the recognition and the fusion of the viral particle with its target cell, this is due to the interaction between the HIV-1 glycoprotein gp120 with the host surface receptor CD4, which is the primary receptor expressed on the surface of lymphocytes, macrophages, monocytes and dendritic cells. HIV-1 also engages a co-receptor for its entry: a CC-chemokine receptor. It usually uses CCR5 and CXCR4. These co-receptors are differentially expressed among the T cells: in particular CXCR4 is expressed by memory T lymphocytes and naïve T lymphocytes, whereas CCR5 is expressed only by memory T
lymphocytes but it is expressed also by macrophages and dendritic cells. Different HIV-1 variants use one or the other chemokine receptor, some can use either. (Deeks S.G., 2015)
The fusion of the viral particles follows the interaction with the target cells. Once inside the cell the capsid is uncoated releasing the ssRNA genome and HIV-1 enzymes, such as RT and IN into the cytoplasm.
At this point the ssRNA is retrotranscribed by the viral RT to dsDNA, known as the complementary DNA (cDNA). The cDNA will form the pre-integration complex (PIC) using both host and viral proteins. This complex is needed to import the cDNA into the nucleus and to facilitate its integration. PIC contains the viral IN necessary for HIV integration and other proteins carrying a nuclear import signals such as IN, MA and Vrp (Sherman M.P., 2002). PIC is imported into the nucleus interacting with the nuclear pore complex (NPC) and gaining the access to the nucleoplasm. This complex is the reason why HIV can infect also non-dividing cells: its genome can reach the host one because it can actively cross the nuclear membrane (Archin N.M., 2014), whereas non-lentiviral retroviruses need the nuclear membrane to dissolve before they can integrate into the host genome.
When the cDNA and the IN are settled into the nucleus the IN can use the HIV-1 cDNA and integrates it into the host genome. The viral IN possesses two catalytic activities: 3’ processing and DNA strand transfer. Each end of the HIV-1 DNA long terminal repeats (LTRs) is cleaved adjacent to the invariant dinucleotide sequence d(C-A), unveiling recessed 3’ termini. IN then uses the 3’-hydroxyls to cut chromosomal DNA strands across a major groove while joining the viral DNA ends to the target DNA 5’-phosphates. Then host
enzymes complete the integration using the host repairing machinery (Engelman A., 2012).
Once HIV is integrated the late phase of the viral cycle can start. Integrated HIV is defined as the provirus. The HIV provirus uses the host RNA polymerase II to produce different mRNA strands. Transcripts are exported in the cytoplasm in two different ways; the two pathways are illustrated in figure 3. Unspliced and incompletely spliced HIV-1 RNAs are exported via Rev-dependent export pathway, whereas completely spiced mRNAs, or multiply-spliced mRNAs, exit the nucleus via the normal mRNA export route (Sundquist W.I., 2012). HIV-1 multiply-spliced transcripts are produced also before integration in the early phase, in this case HIV-1 mRNAs are exported into the cytoplasm independently by Rev. With this mechanism mRNAs interact with NxtI receptor, one of the two major receptor implicated in the mRNA pathways
across the nuclear pore complex (NPC). This type of mRNAs encodes for two regulatory proteins, Tat and Rev, and one accessory protein, Nef. The Rev protein is encoded in the cytoplasm and then localized into the nucleus where it provides the other mechanism of mRNA export that occurs in the late phase: the Rev and Rev-Responsive-Element (RRE) dependent one. Into the nucleus Rev can bind HIV-1 mRNA via the RRE, a 221-nt element with six stem loop. Rev proteins multimerize on RRE and interact with Crm1 protein, the second receptor used in the mRNA export across NPC (Okamura M., 2015).
Figure 3 mRNA transport from nucleus to cytoplasm. Differences between early and late phase of HIV infection (Okamura, 2015).
Viral RNAs located in cytoplasm can be used as viral genome or to produce long chains of HIV-1 precursor proteins, then cleaved into functional ones. The Gag polyprotein precursor is proteolysed by the viral protease (PR) to generate the Gag proteins matrix (MA or p17), capsid (CP or p24), nucleocapsid (NC or p7) and p6 proteins. Pol encodes for the Gag-Pol polyprotein precursor that is processed by PR releasing protease (PR), reverse transcriptase (RT) and integrase (IN). The env gene encodes for a precursor protein that is cleaved by host factors in a 30-amino-acids signal peptide (SP), gp120 and gp41 (Watts J.M., 2009).
When ssRNA dimers and HIV-1 proteins are released into the cytoplasm they interact with the inner leaflet of the plasma membrane, where they are assembled, and with each other in order to be packaged into a new infectious particle.
The assembly is drove by the Gag and Gag polyprotein (Gag-Pro-Pol), synthetized from the GagPol precursor and then translocated to the site of assembly. HIV-1 Gag mediates all of the essential events in virion assembly, it helps in binding the plasma membrane, in protein-protein interactions, Env recruitment and the packaging of the HIV-1 RNA genome.
Gag polyprotein is composed by different domains, starting from the amino-terminal domain there is MA domain, the central domain is CA, then the NC domain flanked by spacer peptides SP1 and SP2, and P6 as the carboxy-terminal domain (Sundquist W.I., 2012).
The HIV Gag and Gag-Pro-Pol polyproteins move from their site of synthesis in the cytoplasm to the plasma membrane. When Gag reaches the plasma membrane, it interacts with the MA domain by the recognition of a specific phospholipid, PtdIns(4,5)P2, that is associated to lipid rafts. In fact viral production is strictly associated with these regions and the virus envelope is not only characterised by Env but also enriched in raft-associated proteins and lipids. Env glycoproteins, gp41 and gp120, and the accessory protein Vpu encoded by the same RNA are translated on the rough ER (Sundquist W.I., 2012). Env proteins reach the plasma membrane and accumulate at the site interacting with the MA domain of Gag, then gp41 anchors the gp120/gp41 complex in the membrane as shown in the figure 4.
The NC Gag domain is important in the packaging of the functional dimeric unit of HIV ssRNAs. NC contains two retroviral zinc finger domains and acts like a chaperon in the interaction with the RNA dimer. The two RNAs start their non-covalently interaction with a loop formations at their 5’UTR, further loops are formed from this interaction. Efficient genome packaging depends on the ψ-site, a sequence that comprises four stable stem loop structures, which interacts with the two NC zinc finger domains.
In addition to the viral genome, also tRNAs molecules necessary for the initiation of reverse transcription are required to be packaged in the virion. Two copies of tRNA anneal via Watson-Crick base pairing to the primer binding site (PBS), a different loop in the RNA genome (Sundquist W.I., 2012).
Figure 4 HIV-1 assembly during the late phase of replication cycle (Freed, 2015).
Once Gag polyproteins, Env, and the viral genome are assembled as a complex to the membrane the nascent viral particle must undergo a scission event to be released from the cell, this event in named viral budding (Freed E.O., 2015). In this phase the virion crosses the plasma membrane and obtains its lipid envelope. The budding vesicle takes place when Gag level, that increases exponentially, reaches a plateau. This process is mediated also by the host endosomal sorting complexes required for transport (ESCRT) machinery, which has a rule in vesicle budding, cytokinesis and virus release. A kinetic study for the ESCRT recruitment and the HIV-1 assembly and release showed that the kinetics of assembly has an average of 5-9 minutes and the ESCRT complex is required in the final step of the particle release and it is necessary in driving membrane scission.
After budding the virion is released but it is still an immature and non-infectious particle and it needs to undergo a process of maturation before it can be capable of infection. In the immature particle Gag molecules are packed radially, with their amino-terminal MA domains bound to the inner membrane leaflet and the P6 facing the interior of the particle. Important changes that occur during HIV-1 maturation include activation of the fusogenic activity of the
viral Env protein, capsid assembly and stabilisation of the genomic RNA dimer and
rearrangement of the tRNALys,3 primer-genome complex (Sundquist W.I., 2012).
Viral maturation, so the gaining of the particle infectivity, requires the activity of the viral protease (PR). PR cleaves a number of sites in both Gag and Gag-Pol polyproteins, producing the fully processed MA, CA, NC, p6, PR, RT and IN proteins. This step generates a great change in the virion morphology, after the releasing of the individual proteic domains HIV-1 capsid form the conical capsid core called “fullerene cone”, specific of the infective viral particle (Freed E.O., 2015).
2.4 Infection
HIV-1 infects CD4+ T-cells and monocyte-macrophage lineage cells, involved in immune responses, developing quantitative and qualitative defects in CD4+ T-cells. HIV is transmitted through contact of infected body fluids with mucosal tissue, blood or broken skin, this happens via vertical transmission (mother-foetus), men having sex with men (MSM), sexual intercourse, injection of intravenous drugs, and infected blood transfusions (Lucas S., 2015). After the transmission event, the viral infection starts. HIV-1 infection can be divided in three phases: a primary or acute infection characterised by a spike in the viral load followed by a chronic phase, where the viral load is more stable, and at last by the arising of AIDS.
2.4.1 Primary Infection
The primary infection starts from the moment of the viral transmission and last until the 9th
week. This phase is characterised by the rising of the viral load, consequently the virus is detectable at high level in blood, and by an initial depletion of CD4+ T-cells as shown in figure 4 and figure 5. The first days of the acute infection establish the viral reservoirs, which is the main barrier to the cure.
In the last part of primary infection patients can show some flu-like symptoms. A transient illness resembling infectious mononucleosis can appear one to twelve weeks after exposure. Primary HIV-1 infection symptoms can be: fever, lymphadenopathy, pharyngitis, arthralgia, myalgia, rash, lethargy and occasionally aseptic meningitis. Symptoms persist one to two weeks. The severity varies dramatically. Some manifests no symptoms whereas others can develop serious manifestations such as candida esophagitis (Wormser G., 2004).
Figure 5 A) Plasma HIV-1 RNA levels during all the phases of HIV-1 infection. B) CD4+ T-cell count during the course of HIV-1 infection (Deeks SG, 2015).
Exposure to HIV-1 is primarily through the mucosal route, either gastrointestinal or reproductive. After the transmission event we assist to the infection of the first target cells in the mucosal tissues at the site of infection, and within days HIV-1 spreads to draining lymph nodes and eventually to distal lymph nodes and other tissues. Studies on primates inoculated with SIV indicate that the first viral substrate is CD4+ T-cells in lamina propria of the mucosal tissues, rather than non-lymphocyte targets such as dendritic cells. Despite dendritic cells are target for HIV-1, but not the initial target for viral replication, they seems to be relevant as local facilitators for productive infection (Douek D.C., 2003). Due to the viral spreading, HIV-1 can accumulate to high levels in the pool of lymph node follicular dendritic cells (FDC) during acute infection and these cells may become a major reservoir of infectious HIV-1 in the late stages of infection (Douek D.C., 2003).
In acute infection, at about day 10, viral particles start to be detectable in the blood and rise exponentially over the next few weeks, usually peaking at day 30-60, when the HIV-1 RNA
at plasma level can reach a concentration of 106-107 copies per ml, as depicted in figure 4A.
This concentration is different among patients (Deeks S.G., 2015). Assays for the HIV-1 Gag protein p24 in the serum are typically positive, reflecting high levels of free virus in circulation (Wormser G., 2004). Another consequence of the high viral title is that individuals are probably most infectious at this point. On the other side during primary infection there is a loss of CD4+ T-cells from week 0 to 6 as shown in figure 4B. Week 6 is the moment when the immune system starts its response against the infection bringing to a raise in CD4+ T-cell concentration. CD8+ lymphocytosis during primary infection perhaps reflects a cytotoxic T cell (CTL) response to the virus (Wormser G., 2004). HIV-1 specific CTL response can be detected in the first weeks after infection, contributing to the control of acute HIV-1 infection.
As the immune response to HIV-1 develops around week 6, there is a dramatic reduction in viremia and CD4+ T-cells count starts to increase reaching a plateau into normal level range. Typical set point levels are from 10.000 to 100.000 RNA copies per ml of plasma. The plateau level is relevant, because it determines the rate of disease progression; a lower level means a slower evolution of the disease and vice versa. The plateau reflects the balance between viral replication and host immune responses, main factors in the second phase of infection where, over time, the virus causes a slow depletion of CD4+ T-cells (Wormser G., 2004). Another serious event during the acute infection consists in a very deep impact on the gastrointestinal tract, which may be the largest lymphoid organ in the body: it has been estimated that it contains at least 60% of the total body T cell load. The epithelia of the gastrointestinal and respiratory tracts are the major site for the sequestration of memory CD4+ and CD8+ T-cells tissues, expressing CCR5, and are prime targets for R5-tropic HIV-1 infection and replication. During the acute phase much of the HIV-1 replication takes place in gut-associated lymphoid tissue (GALT), producing a severe T-cell depletion that is thought to make the intestinal lining permeable, causing a systemic translocation of bacterial products that leads to increased immune activation (Deeks S.G., 2015).
2.4.2 Chronic Infection
The second phase of HIV-1 infection is named chronic infection; this phase can last for yeas and is the long asymptomatic period between primary infection and the development of AIDS. It is characterised by a constant HIV-1 replication that remains relatively stable, without any manifestation of symptoms, as a consequence there is a slow increase in the viral load until a set point as shown in figure 4A. Complementarily, there is a gradual depletion of CD4+ T-cells, level of CD4+ T-cells may be initially be stable but inevitably begins to drop. If the CD4+ T-cell count is the best predictor of clinically significant immunodeficiency, the level of viremia, HIV-1 RNAs in plasma, determines the rate at which cells are lost, suggesting that viral infection is linked to CD4+ T-cell destruction, however this mechanism is not totally clear; CD4+ T-cell depletion is examined later.
The CD4+ T-cell fall is generally gradual with a more rapid drop in the 18 months preceding the development of AIDS (Wormser G., 2004). The average time in adult to develop AIDS is from 10 to 11 years, on the extremes at about the 20% of the patients develops AIDS within 5 years of infection and the 12% can remain free of the AIDS for 20 years. HIV-1 RNA plasma level is a good marker in predicting the development of AIDS with a strong time-dependent correlation between the HIV-1 RNA concentration and the outcome (Mellors J. W., 1996). On the other side patients following antiretroviral therapy show low HIV-1 RNA plasma level and usually do not develop AIDS, otherwise they can develop different complications due to continue assumption of these drugs.
As said above, the major co-receptors used by HIV-1 in vivo are CCR5 and CXCR4, these co-receptors are differentially expressed among the T cells and different HIV-1 variants use one or the other chemokine receptor, some can use either. New infections are typically established by one or few genetic variants (Deeks S.G., 2015). CCR5 expressing cells are more abundant at a mucosal level whereas CXCR4 is higher in CD4+ T-cells in peripheral blood and lymph node. CCR5 is usually the initial target coreceptor for naturally transmitted virus, thus CCR5 tropic; in this case a CXCR4 tropism develops over time in many individuals, due to the high mutation rate of the virus that becomes capable of expanding its target cell repertoire. Symmetrically, if the infection starts from a virus that targets CXCR4 a CCR5 tropism can evolve in the course of infection. Consequently, the dynamics of CD4+ T-cell loss varies between the two. In 1999 a study by Harouse’s team demonstrated this association between the HIV-1 tropism and the dynamics of the cells depletion. Harouse and
collaborators have used chimeric viruses (SHIVs) composed by a SIV backbone and the HIV-1 envelope. The HIV-1 envelope were designed to be R5 or X4 tropic. As expected, following the infection R5 tropic SHIV infection resulted in a deep depletion of intestinal CD4+ cells whereas infection with X4 tropic virus produced a greater depletion in CD4+ T-cells in peripheral blood and lymph nodes (Douek D.C., 2003).
CD4+ T-cell depletion has multifactorial causes including impairment of lymph nodes stability and cytotoxicity. Importantly, studies suggest that in chronic phase only a small fraction of CD4+ T-cells are infected and sustains an active viral replication cycle. Analysis of the fraction of CD4+ T-cells from patients shown that most of the detectable viral DNA is an unintegrated form and integrated virus is found in about 0.01% of CD4+ T-cells and macrophages (Wormser G., 2004), elucidating the fact that CD4+ T-cells harbouring replication competent provirus are rare. In addition, the extent of infection is higher in the lymph nodes than in the peripheral blood. Lymphocytes find themselves almost in lymph nodes. The ones in circulation represent only the 2%, of the total population, during the asymptomatic period of the infection the proportion of cells carrying HIV-1 DNA is higher up to 10 folds in the lymph node that in the peripheral blood indicating that lymph node is mostly associated with the spreading of infection. Virus particles in the germinal centers of the lymph nodes have been found associated with the network of follicular dendritic cells (FDCs). FDCs are normally involved in a complex network in lymph nodes and they function as a filter that traps viral particles reducing viremia. FDCs are antigen-presenting cells capable of binding antigens that have bound antibody and/or activate the complement system. These cells interact also with B-lymphocytes activating them in response to the antigen. During the asymptomatic phase in the chronic infection there is a progressive invalidation of the lymph nodes architecture, including loss of FDC network. This loss is responsible of the abnormal B cell function during HIV-1 infection and of the raise in viral load. In addition, FDCs bind virus particle on the membrane thus infecting CD4+ T-cell migrating in the lymph nodes; this mechanism occurs also in blood dendritic cells (Wormser G., 2004).
2.4.3 CD4+ T-cells depletion
During the primary infection a transient reduction of CD4+ T-cell count in blood is detected, anyway these cells rebound to near-normal levels in the end of acute infection; later, in the chronic infection, CD4+ T-cell count slowly declines both in tissues and in blood. It has to be
taken in account that, as previously explained, the dynamics of how the CD4+ T-cells are lost varies if the infectious virus is R5 or X4 tropic.
During infection much of the HIV-1 replication and CD4+ T-cell death occurs in lymphoid tissue, particularly in lymph nodes and in gut-associated lymphoid tissue (GALT), which harbours high numbers of susceptible memory T lymphocytes. Furthermore, HIV-1 accumulates to very high levels in FDCs. Lymphoid tissues are fundamental for the infection because they harbour HIV-1 target cells in elevated density, providing a continuum of targets, which is relevant because cell-to-cell transmission of virus is a local phenomenon, an infected cell releases virus that efficiently infects only nearby targets (Douek D.C., 2003). The profound CD4+ T-cell depletion in the acute phase leads to an activation of the immune system with high turnover of lymphocytes in these compartments, that partially regenerates depleted mucosal compartments and prevents early onset of immune deficiency. In the beginning of the chronic phase there is a condition of equilibrium between cells depletion and immune activation, in some cases the CD4+ T-cell level can be stable during the first phase of chronic infection, but it will inevitably leads to the gradual CD4+ T-cell loss of the chronic infection and to the AIDS onset.
CD4+ T-cell depletion can originates both by directed viral toxicity and by indirect viral toxicity, but considering the fact that T-cell depletion is a slow process while viral replication is continuous during almost the chronic phase of infection and because of only 0.01% of CD4+ T-cells is infected makes logical that lymphopenia is not only the result of viral directed cytophacity. In fact with the simian immunodeficiency virus (SIV) infections of African non-human primates (NHPs), from which HIV originates, are largely non-pathogenic. The SIV infection in natural hosts is just as cytopathic to NHP CD4+ T cells as HIV is to human CD4+ T cells, but in the vast majority of these animals, CD4+ T-cell depletion is not progressive and AIDS does not ensue (Okoye A.A., 2013). Also, the HIV-2 subtype, which is more similar to SIV than HIV-1, is less pathogenic than the latter and its infection produces a lower CD4+ T-cell depletion. Therefore CD4+ T-cell depletion in the chronic phase is mainly due to the virus-induced alteration of the equilibrium of the immune system, with anomalous cell kinetics in their compartments and the progressive impairment of thymus and bone marrow functionality, while direct and indirect viral cytopathogenicity play a minor role in the lymphopenia.
For what concern direct HIV toxicity, HIV-1 can produce CD4+ T-cell depletion in different ways: creating syncytia or with viral replication cycle products that are toxic to the cell. Syncytia, or multinucleated giant cells, are formed by the fusion of infected cells expressing Env glycoprotein and non-infected cells expressing CD4; these are short-lived structures. Inclusion of non-infected CD4+ T-cells in syncytia provides a potential mechanism for CD4+ T-cells depletion. The extent to which syncytium formation contributes to depletion is not clear because giant cells have been rarely observed but this could also means that syncytia are a labile structure. Infected cells die independently of any cell-cell fusion events because of viral replication and incomplete reverse transcripts products stimulate an intense inflammatory response or apoptosis. It has been seen that many cell types poorly tolerate Env glycoprotein and that susceptibility is related to levels of CD4 expression, perhaps reflecting toxic consequences of intracellular interaction between Env and CD4. Conversely HIV has evolved mechanisms to control this toxicity enhancing an endocytosis mechanism of Env in excess (Wormser G., 2004). Other HIV proteins including Tat and Vpr have also been implicated in death of infected cells. Different data reports that HIV primes apoptosis of CD4+ and CD8+ T-cell. Apoptosis is a process of programmed cell death morphologically characterised by blebbing, cytoplasm and nucleus condensation and chromatin fragmentation and biochemically by the activation of the proteases family of the caspases. By 1995, it had been established that HIV apoptosis is stimulated via Fas (CD95/APO-1) receptor, a member of the tumour necrosis factor receptor (TNFR). Data shows that the proportion of lymphocytes expressing Fas is more elevated in HIV-infected individuals, and the proportion of Fas-expressing cells increases with disease progression. Analysis on lymph node biopsies detected many apoptotic nuclei, importantly a study by Finkel et al. shown that few nuclei contained viral RNA- and that the predominant cellular population was not the CD4+ expressing one, indicating that apoptosis is an indirect effect of HIV infected cells on un-infected “bystander” cells (Jaworowski A., 1999).
An indirect toxic effect involves the natural immune response to HIV-1 infection from CD8+ cytotoxic T-lymphocytes (CTL) mediated destruction of infected cells. Most of the current evidence suggests that CTL plays a role in lysis of infected cells (Wormser G., 2004).
Although these mechanisms have been observed, they cannot be the only reason of the profound CD4+ T-cell depletion because of the low frequency of infected cells. HIV-1 infection contributes to a global deregulation of T cell dynamics, affecting the turnover of both infected and uninfected cells. As outlined before, the systems most affected by depletion
are GALT and lymph nodes. For what concerns GALT, there is a strong depletion of memory T-cells that occurs in the acute phase of the infection during the first 2-3 weeks, this event reflects its effect during the chronic phase. As mucosal immune system, GALT is specialized for antigen uptake, transport, processing and presentation, for the production of distinctive immune effector cells and a specific homing program. Mucosal tissues act as primary lymphoid sites where lymphocytes develop from immature cells and differentiate into effectors cells. A study with intravenously applied simian immunodeficiency virus (SIV) confirms this lymphopenia, as shown in figure 6, and reported that this loss is restricted to
terminally differentiated CD4+ TEM, while leaving relatively intact the precursor CD4+ TCM
population that does not express CCR5, capable of continuous high level production of new
TEM (Okoye A.A., 2013). This loss of lymphocytes has been attributed both to direct infection
and to Fas-Fas-ligand induced apoptosis, as reflected by a gp120-induced increase of Fas ligand mRNA (Kraehenbuhl J-P, 1998).
In any case, this large depletion of T memory cells occurs in the acute phase of infection, long
before the onset of AIDS. But considering that the virus leaves intact the CD4+ TCM cellsit
assures itself of a continuous supply of new targets. This would suggest that the profound depletion of CCR5+ CD4+ memory T cells during acute infection would not lead to AIDS progression if the regenerative potential of these could be effectively maintained over the long term. Indeed, this seems to be the case with SIV infections in NHPs, where there is no profound CD4+ T-cell depletion and no induced alteration in the cell turnover as in HIV-1
infection (Okoye A.A., 2013). TCM cells decline at GALT level will impede the maintaining
of CD4+ TEM cell production in time, leading to the outcome of the disease, an observation
that sustains this fact came from studies on primates using SIV, which shown that regeneration produces short-lived cells, whereas in healthy individuals proliferating cells are predominantly long-lived. Short-lived cell production necessitates higher levels of proliferation to maintain the homeostasis at subnormal levels and not all functionally important CD4+ subsets are regenerated to the same degree (Okoye A.A., 2013).
Also lymphnodes play an important rule during the infection. The rapid rise in blood CD4+ T Cell counts in the final stage of acute infection probably derived from redistribution of T cells from the lymph nodes into the blood. The majority of CD4+ T-cells resides in the lymph nodes, providing an environment with high density of target cell necessary for the spreading of infection, and only 2% of the population is in the blood. As said before, virus particles in
the germinal centers of the lymph nodes have been found associated with FDC, producing an extension of the infection and an abnormal B cell functionality.
Figure 6 CCR5 CD4+ T-cell depletion in SIV infection. Only differentiated CD4+ TEM cells are depleted, CD4+ TCM cell population is intacted but it will provide future target TEM cells for SIV (Okoye AA, 2013).
HIV infection influences the capacity to form new cells that affects bone marrow and thymus, inducing a high CD4+ T-cell turnover; the rate of cell loss exceeds the rate at which these are produced through thymic differentiation and/or clonal expansion of peripheral CD4. Thymus is the primary site of T-lymphocytes development and HIV-1 infection of the thymus can have a profound impact on thymopoiesis. HIV-1 infection changes thymus morphology, accelerating its involution that normally occurs with age but the mechanism is still unclear. The importance of thymopoiesis in regeneration of peripheral T cells is dependent on both the age and immune status of the patient. Adults display a reduced capacity for cell regeneration than adolescents and thymic-dependent pathways of maturation reactivation in adults is uncertain, contrarily to the younger patients. Therefore, it appears that disease progression in adults is more linked to a loss of peripheral, thymic-independent regenerative capacity (Gaulton N.G., 1997).
2.5 Reservoirs
HIV-1 can establish either a productive infection or a non–productive one, named as latent infection. Viral latency is defined as a reversibly state of non-productive infection of the individual cell, which became a reservoir. Specifically, an HIV-1 reservoir is defined as a cell
type or anatomical site that allows persistence of replication-competent HIV-1 for long periods of time in patients on optimal HAART regimens. In a practical sense, reservoirs are barriers to eradicate in order to provide a definitive cure for HIV infected patients. (Siciliano R. W., 2011)
During HIV infection latency can occur at preintegration and the postintegration phases. In the first one the HIV genome is maintained non-integrated and silenced in the cytoplasm; the second one develops when the provirus is integrated in the host genome but it is not expressed. This form is the postintegration latency, is the most relevant and is described first.
2.5.1 Postintegration latency
The first evidence that CD4+ T-cells with integrated provirus can function as a latent reservoir for HIV-1 infected individuals was reported in 1995, before the wide application of combinatorial antiretroviral therapy (cART) begun. In these cells viral expression is repressed, but the virus maintains the ability to reinitiate its replication cycle (Chun T.W., 1995). The first indication that true eradication of HIV-1 in infected individual would be extremely hard came in 1997, with the publication of three independent studies that demonstrated the persistence of a latent and inducible viral reservoir in all study subjects, despite the ART had maintained their plasma viremia below the limit of detection for a few years.
HIV-1 establishes latent infection mainly in memory CD4+ T-cells. As said above, an important source of source of CD4+ T-cells depletion derives from the gut-associated lymphoid tissue (GALT) in the primary infection. GALT in this phase of infection is also a consistent origin of latently infected CD4+ T-memory cells. Latent infection can also occur in naïve CD4+ T-cells, cells of the monocyte and macrophage lineage, and perhaps in other long-lived cells (Deeks S.G., 2015). The latency establishment occurs very early in infection, it has been shown that cART treatment after 10 days from infection did not prevent the establishment of a pool of latently infected CD4+ T-cells (Chun T.W., 2012).
Latency occurs in resting cells, but HIV-1 can not efficiently establish a productive infection in a resting CD4+ T-cell because it needs several host transcription factor to integrate that are not available in resting cells. There are two hypotheses to explain how a latent infection can be established as shown in figure 7. A first one sustains that, in response to an antigen, resting
CD4+ T-cells undergo a burst of cellular proliferation and differentiation. Most of effector
cells die but a subset survives, rests in a G0 state and persists as memory cells. A second
hypothesis asserts that latently infected memory cells can derive from an infected activated CD4+ T cell that have reverted back to a resting state (Siciliano R. W., 2011).
However, resting CD4+ T-cells represent a very small fraction of all cells. They persist in vivo because of their memory phenotype and because they don’t express viral antigens on the surface so they are not recognised by the immune system (Marcello A., 2006). Their homeostatic reactivation and proliferation contributes to a new infectious event and to the maintenance of the viral reservoir (Okoye A. A., 2013).
Figure 7 HIV-1 postintegration latency. Latency establishes when active CD4+ T-cells become infected and revert in a memory state, which are characterised by a long life span (Siciliano R.W., 2011).
In addition to CD4+ T-memory cells, other cellular types can play a rule as reservoirs. HIV-1 infects also cells of the monocyte-macrophage lineage. Infected macrophages may constitute a particularly important source of virus late in the course of disease. There are evidences for infection of circulating monocytes but these cannot be considered a reservoir because they circulate only 1 day before they differentiate in macrophages, but on the other hand monocytes can permit the virus an entry into the central nervous system (CNS), they can cross the blood-brain barrier and differentiate into macrophages and microglial cells. These
cells represent a viral reservoir and CNS pathology in HIV-1 infection, and there is evidence in the SIV model for latent infection (Siciliano R. W., 2011).
Latent cells have integrated the viral sequence but this is not expressed, in other words, viral genome is not transcribed and thus not translated. Because of the provirus is integrated into a eukaryotic genome, HIV-1 gene transcriptional activation or silencing will respond to the same factors of the host genome: gene expression could be repressed either by chromatin condensation or by transcriptional interference. Other factors that contributes to HIV transcription are paucity of cellular factors required for robust HIV expression in infected resting cells, interference of HIV expression by the proximal host gene promotes.
Chromatin architecture plays a relevant rule in gene regulation; chromatin is divided in two different conformations: euchromatin and heterochromatin. Euchromatin is a relaxed form that permits transcription factors to interact with the double helix, thus permitting gene transcription; contrarily heterochromatin is a condensed state that impedes access of transcription factors to DNA, inhibiting transcription.
Data from the J-lat cell line latency model, and from patients CD4+ t-cells revealed that HIV-1 can integrate not only in heterochromatin but also in euchromatin regions, in other words is genes that are normally expressed in an active cell. It has been found that HIV-1 is able to stimulate chromatin architecture into a condensed heterochromatin state. HIV-1 LTRs in the provirus are associated to two nucleosomes, which can modulate nuc-0 and nuc-1. These can modulate proviral expression; nuc-1 in particular is linked to activation or silencing of viral genes. One or more histone deacetylases (HDAC) are active on the LTR of HIV-1 where nuc-1 is maintained, supporting the inactivation of proviral genes. HIV-nuc-1 enhances the nuc-nuc-1 maintenance recruiting HDAC-1 through its interaction with multiple host factors. Proviral condensed state is also helped by mutilation of two CpG islands that flank HIV-1 transcription start site, it has been found that in a latent cell HDAC2 is associated to this region (Siciliano R. W., 2011).
2.5.2 Preintegration latency
During HIV-1 infection not all the cDNA is integrated in the host genome, in fact, the majority of viral DNA remains unintegrated in HIV-1 infection both in vitro and in vivo,
regardless of cell type and activation status. In blood, lymphoid tissue and brain unintegrated DNA (uDNA) is up to 100 times more abundant than integrated DNA (iDNA). uDNA accumulates in both activated and quiescent T lymphocytes, in monocytes and in patients with high or low viral loads. Importantly, both linear and circular unintegrated DNA is transcriptionally active (Huub C. G., 2008). The uDNA is normally produced and sustained during the course of infection even in the absence of detectable plasma viremia, thus their presence may indicate either continuous cryptic viral replication or a persistent viral structure in a non-dividing long-lived reservoir. In this sense, in some circumstances, they can be considered as viral reservoir.
2.5.2.1 Conformations of unintegrated viral DNA
uDNA has been found in different conformations. The most abundant form is the linear cDNA that is the direct product of reverse transcription and the substrate of the integration. Linear DNA is rapidly degraded because it is toxic to the cell; an excess of linear dsDNA induces an apoptotic response. All the others conformations have been found in a circular structure (Sloan D.R., 2011); circular unintegrated HIVs formation is an important stratagem to avoid cellular apoptosis. Double-strand ends of the viral cDNA provide the pro-apoptotic signal, they are the normal substrate of the NHEJ pathway and they are removed being transformed in cDNA. These findings extend an observation made by Howard Termin 20 years ago that the amount of unintegrated viral cDNA in infected cells correlates with the cytopathicity of retroviral infection (Ling L., 2001).
As shown in figure 8, circular ectopic HIV-1 can be obtained with three different mechanisms: autointegration, recombination and host DNA repair.
The autointegration, also called suicidal integration, is a suicidal pathway that aborts the infection. Autointegration occurs because of the IN activity, which processes the 3’-end of reverse transcript to integrate the provirus in the classic infection but in this case IN ligates viral ends together, producing either internally rearranged or less than full-length circles.
Another option is the formation of 1-LTR circles that are circular structures characterised by a viral sequence carrying only one LTR. These are formed through host DNA repair machinery operating in the Homologous Recombination (HR) at the viral regions of the LTRs that have a homologous sequence. Consequently the resulting circle bears only one of the two viral LTR.
1-LTR circles have been found exclusively in the nucleus because the formation of these structures requires the cellular nuclear factors involved in the HR such as RD50/MRE11/NBS1 proteins.
Figure 8 Different conformation of HIV-1 uDNA. HIV-1 cDNA if not integrated can assume a diffenent circular structure via different mechanisms, producing autointegration products, 1-LTR and 2-LTR circle structure (Sloan R. D., 2011).
On the other side 2-LTR circles derive from the host DNA repair machinery involved in the non-homologues ends joining (NHEJ) as a response of the cell to linear DNA. Consequently, also these structures have only been found in the nucleus too. This is confirmed because it has been shown that the Pre-Integration Complex (PIC) is associated with the protein Ku, a protein involved in the NHEJ mechanism, and that the inactivation of host NHEJ factors such as Ku, Ligase4 or XRCC4 leads to a reduction of the 2-LTR circles.
At last, 2-LTR circles can be easily monitored by PCR because they can be read thanks to the double copies of LTR sequence and it has been seen that 2-LTR circles is the less abundant species among the circular unintegrated HIVs (Sloan D.R., 2011).
2.5.2.2. Stability and persistence of unintegrated HIVs
If linear unintegrated DNA is rapidly degraded, on the other hand, circular unintegrated DNA is highly stable, it can persist for long time into the cell and therefore could be considered as a reservoir. In previous years unintegrated DNA was considered non-stable and rapidly degraded, and consequently it has been proposed that it could have been used as a marker for ongoing infection (Scott L. B., 2002).
But, as said above, circular unintegrated DNA is highly stable, anyway, lacking an own origin of replication, it is not copied during the cell division and it could be lost only through cell death, dilution during cell division, or degraded. Because of the rapid rate of active lymphocyte turnover unintegrated circular HIVs do not persist for a long time. On the contrary they can persist longer in non-diving or slow-diving cells such as quiescent, memory, CD4+ T-cells. In macrophages, which normally do not divide, it has been seen that uDNA can persist and remain transcriptionally active for at least 30 days (Huub C. G., 2008).
2-LTR circles have been considered for the study of circular HIV persistence in a quantitative study from Scott L. Butler. This work shows that total HIV uDNA accumulated quickly after infection, reaching maximum abundance after 12 to 24 h. Initially this corresponds to linear double strand viral DNA. The 2-LTR circles and integrated forms accumulated slower, reaching maximal abundance after 24 h. The final number of provirus per cell is considerably lower than the total number of cDNA copies, only a fraction of the HIV DNA is converted to proviruses. After 24 to 48 h, all of the viral cDNA is in a proviral or circular form (Scott L. B., 2002). Importantly, treatment with cART can increase the number of 2-LTR circles: intensification with raltegravir, an integrase inhibitor, led to a transient increase in the level of 2-LTR circles (Laskey S.B., 2014).
2.5.2.3 Transcription and Translation of unintegrated HIVs
All unintegrated circular HIVs forms have been found to be transcriptionally active. Integrase mutant or defective viruses have been used to evaluate and characterize the transcriptional activity of unintegrated circular HIV, there is difference in the transcriptional activity due to the mutation type or cell type used. Also integrase inhibitors such as raltegravir can be used to block integration and study uDNA activity. All three species of uDNA could serve as transcriptional template, albeit at a lower level than the integrated provirus. The lower transcriptional ability of uDNA compared to the integrated form might be due to the
proximity of the two LTR promoters or the use of the single LTR to both start and end transcription in the 2-LTR and in 1-LTR circular forms (Cara A., 2006). Another reason of the low transcriptional capacity of uDNA in certain cell types suggests that the structural conformation of this DNA does not make it an efficient template for viral expression. Proteins of either cellular or viral origin might impede transcription or, on the contrary, promote it. For example the presence of HIV-1 Vpr protein is essential for optimal expression of unintegrated viral templates. It has been shown that virally imported Vpr can promote the transcription of viral genes from uDNA, independently of Tat transactivation. This process of Vpr mediated transcription may ultimately lead to Tat expression and subsequently provide a positive feedback of the transcription process from uDNA via Tat (Sloan R. D., 2011).
For what concern transcription of viral genes from uDNA, this produces all classes of mRNAs: multiply-spliced, singly spliced and un-spliced. The relative proportions of each spliced class vary compared to those observed during productive infection. Multiply-spliced transcripts are abundant in the absence of integration, levels of singly-spliced and un-spliced transcripts are reduced in this circumstance. Both integrating and non-integrating virus produced similar levels of multiply spliced viral mRNA transcripts, and pre-integrated viral transcription is similar to circular viral DNA one (Sloan R. D., 2011). In fact, as explained in HIV-life cycle (section 1.3), in the early phase of infection when the cDNA is not yet integrated only multiply-spliced transcripts are produced encoding for early genes and producing Tat, Rev and Nef proteins. Moreover, a study published by Brussel et al. described a transcript unique to the LTR-LTR junction of 2-LTR circles, though it is unknown if this transcript fulfils any function (Brussel A., 2003). A study conducted by Kantor et al. showed the possibility that gene expression of uDNA can be influenced by chromatin structure. In this work SupT1 cell line and macrophages were infected with integration defective virus, and when treated with an HDAC inhibitor, which turns heterochromatin to a relaxed conformation, showed a higher level of viral gene expression (Kantor B., 2009).
Transcription is produced for both early and late genes in all the three forms of uDNA. For what concern translation this involves mostly early genes Tat, Rev and Nef; but late gene translation is stronger when a superinfection or coinfection is established. A key limitation in the translation of viral genes is represented by the paucity of Rev from uDNA, which limits the nuclear export of Rev-Response-element (RRE) bearing singly spliced and un-spliced transcripts which code for structural proteins or are incorporated into nascent virions. 1-LTR
circles in particular could lead to high level of viral protein expression. However, all unintegrated HIV DNA forms yielded levels of protein synthesis that were an order of magnitude led than for integrating virus (Sloan R. D., 2011). In protein expression there is no direct evidence for Tat expression but an undirected one subsists because of the transactivation of LTR by Tat. Also Rev protein is hardly detectable directly. Nef is the only viral protein that has demonstrated to be expressed (Sloan R. D., 2011).
A study by Wu et al. describes different scenarios in which the unintegrated circular viral DNA may contribute to a productive viral replication cycle. In HIV-1 infection is possible to find cells that harbours only unintegrated circular HIV and cell coinfected or superinfection by multiple viral particles as illustrated in figure 9.
In a first scenario we can imagine a cell containing only unintegrated circular HIV, this can be favoured in a situation where integration is restricted. Unintegrated circular DNA cannot produce all viral particles and sustain independent replication per se. It has been seen that it can encode for mostly early proteins such as Tat, Rev and Nef. Both Tat and Nef can modulate T cell activity to facilitate activation, this would expand cellular targets for viral infection. In this way the unintegrated HIV-1 can modulate the cell in an active state in order to be rescued by a second virus. A different hypothesis sustains that the unintegrated virus may prevent secondary infection because Nef could also down-regulate CD4, so prevent superinfection (Wu Y., 2008). In fact, together with Rev, it inhibits viruses from establishes integration, thus superinfection (Sloan R. D., 2011).
A different scenario could arise with a multiple infected environment, such as in lymphoid tissues, where cells are in close proximity with each other and contact between them is large thus favouring the infection and high local viral concentration. If viral density is high not every virus has the chance to integrate, some will remain circularly unintegrated. In this kind of situation, integrated viruses can rescue the unintegrated ones. Importantly, coinfection is a source of viral recombination, which increases the viral diversity and may increase the fitness of the virus. Another fate for the unintegrated HIV is when a superinfected cell does not provide integration for the incoming virus, which could be used to express viral genes and be assembled into virion particles (Wu Y., 2008).
Figure 9 Model of complementation between unintegrated and integrated HIV-1. The first case (A) rapresent cells infected only by unintegrated HIV-1. The second one (B) describe coinfection of a cell, situation that promotes accumulation of uDNA. The third situation (C) illustrate superinfection of a productively infected cell (Wu Y, 2008).
A recent study by Chan et al. observed productive virion production from uDNA infected cells. Contrarily to the Wu study, Chan previously reported that when HIV-1 infects resting T cells several days prior to T cell activation uDNA alone can generates infectious virions. The study focused on the dynamic of infection between uDNA and iDNA. In uDNA genes are regulated differently from iDNA, only uDNA required an interval of several days after infection to obtain responsiveness to activating agents. They observed that 20 days are necessary to uDNA to reach the same expression of iDNA. Only uDNA depends on Vpr for expression in resting T cells. In conclusion de novo virion synthesis from uDNA is possible but it requires a particular set of conditions that is not the majority circumstance in vivo and in any case the virion production order is several-fold less than iDNA (Chan C.N., 2016).
2.6 Therapies
cART consists in the somministration of a cocktail of different drugs that target the virus in different steps in its replication cycle. With this method patients life quality and life span have remarkably improved. Unfortunately HIV-1 latent reservoirs are not affected by cART, their
persistence forces drugs administration for the whole life of patients. Complete HIV-1 removal from the body remains a challenge. Nowadays two promising experimental strategies have been developed to accomplish this challenge: the proviral genome eradication and the latency-reversal in latent cells. Also RNA interference (iRNA) has successfully been used to silence the viral co-receptor CCR5, providing a protective function, or viral genes, but it requires the continuous presence of siRNA, and the gene ablation is never complete. In contrast, a gene-editing (GE) approach permits to provide an irreversible change in the targeted sequence (Manjunath N., 2013).
2.6.1 Antiretroviral Therapy
The ART helps patients in preventing HIV-1 infection of new target cells with remarkable efficacy but has no effect on cells containing a provirus. Reason why an interruption in cART leads to a viral rebound, even in patients who have adhered to suppressive regimes for years: viral reservoirs, cells in a resting state containing the provirus, can shift into an active state and re-initiate productive viral infection. Although ART cannot target the latent reservoir, extensive clinical and experimental evidence suggest that an optimal ART regimen can inhibit most ongoing cycles of HIV-1. Used in combination they are effective in >70% of patients in bringing blood viral load to undetectable level and enabling the blood CD4+ T cell count to rise nearer normal levels (Lucas S., 2015).
Approved antiretroviral drugs for HIV target different proteins involved in the viral life cycle. Different drug classes available nowadays are represented in figure 10. These are Reverse Transcriptase inhibitors, protease inhibitors (PIs), and the more recent classes integrase inhibitors, fusion and entry inhibitors. The Reverse Transcriptase inhibitors are divided in two classes: the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleoside transcriptase inhibitors (NNRTIs).
NRTIs act as competitive inhibitors for the catalytic site of the enzyme RT, impeding the synthesis of the HIV cDNA. The first approved antiretroviral agent, AZT, is a NRTI. Each NRTI is an analogue of a DNA nucleoside or nucleotide. These analogues are accepted from the enzyme as a substrate but they cannot extend the transcription that will stop prematurely. These kinds of drugs, the earlier ones in particular, causes a certain toxicity because they can interact not only with viral RT but also with mitochondrial DNA dependent DNA polymerase gamma.
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have a different mechanism of action. They are non-competitive inhibitors because they act on an allosteric site of RT changing it conformation so its affinity for the substrate.
Protease Inhibitors (PIs) are competitive inhibitors of HIV protease. These drugs do not block the infection, only non-infectious and immature viral particles are produced, the maturation of the viral particle is PR dependent. Drug resistance-mutations can arise because of mutation in the PR sequence, which is favoured by the rate of mutations on the Gag sequence (Looney D.M., 2015).
Figure 10 Antiretroviral drugs, this table indicated the commercial name of the drug, the related class, and possible toxic effects associated whit it (Lucas S. 2015).
Historically the first antiretroviral drug approved by the FDA in 1987 was an antiretroviral monotherapy based on the somministration of the NRTI zidovurine (AZT). AZT monotherapy selected for drug resistance, and leads to the development of several drugs and the switching from monotherapy to combinatorial one, cART. In 1995 the first PI was developed, followed by the approval of the first NNRTI in 1996. Anyway, it was clear that monotherapies favour the insurgence of viral resistance and 1997 saw cART introduction. In vitro studies suggest that optimal three-drug regimens have sufficient inhibitory potential to
completely suppress HIV-1 replication (Laskey S.B., 2015). Typical cART consist in a backbone of two NRTIs and a base of either a PI or a NNRTI (Asa M.M , 2014).
The cART has considerably improved the life-quality and the life span of infected patients. Anyway relevant aspects for the therapy need to be improved, like the early identification of the infection, the access and the continuum to the therapy. Worldwide, nearly 35 million individuals are estimated to be infected with HIV but only 12,9 million have access to antiretrovirals, and a smaller number still are suppressed. Progress, however, has been made: nearly 67% of HIV positive pregnant women receive prophylactic treatment, and it has been shown that this is protective for the new born, and over 2 million people were started on antiretrovirals worldwide (WHO, 2014).
As said above different problems of cART had to be considered: the adherence to the therapy and the risk of developing a resistance, drug toxicity and drug-drug interactions.
Adherence is a critical factor in the success or failure of antiretroviral therapy. Studies indicate that lower than 95% adherence may lower the success rate by up to 20%. Missed doses cause a decline in systemic drug levels to suboptimal concentrations. A suboptimal treatment can lead to a low-level viral replication and select for a drug resistance that is the major barrier to the effective treatment of HIV-1 infection.
A little resistance can go a long way in limiting effective therapy, often requiring more toxic and less tolerable combinations to be effective. The most common forms of resistance are point mutations in target viral proteins, which often inhibit drug binding. Drug resistance can be transmitted with a founding virus or acquired as the result of treatment to prior regimens. Newer drugs are designed to increase the genetic barrier to resistance. In cells that are infected by multiple virions, drug efficacy can be lower despite the absence of resistance mutations.
The majority of toxic effect from cART arises from erroneous doses and, also if in general co-administration of different drugs gives an enhanced repression, from drug-drug interactions or drug interactions with medications due to metabolic antagonism. Overdose is rare, the most serious reports have been reported in neonates who were inadvertently administered supertherapeutic doses of ART drugs (Asa M.M., 2014).
