FUNCTIONALIZED VIRUS-LIKE PARTICLES FOR CATCHING DENDRITC mi-RNAs

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University of Pisa

Course of Molecular and Cellular Biology

Degree thesis

FUNCTIONALIZED VIRUS-LIKE PARTICLES FOR

CATCH-ING DENDRITC mi-RNAs:

Candidate: Ludovico Maggi

Supervisors: Prof. Antonino Cattaneo, Dr. Cristina Di

Primio

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ABSTRACT

In the central nervous system, RNA localization plays a pivotal role since the local translation of mRNAs at dendrites can be triggered by synaptic activation and is thought to induce plastic changes that occur at synapses triggered by learning and memory. There are multiple advantages in mRNA localization as a key regulatory mechanism to fine-tune gene expression. First, the localization of mRNA, rather than its corresponding protein, targets the protein directly to the correct intracellular compartment while preventing its expression elsewhere. Second, it provides a synapse with the unique opportunity to spa-tially restrict gene expression with high temporal resolution. Third, it is more economic to reuse a giv-en transcript several times for multiple rounds of translation instead of transporting each protein or transcript individually to a distinct synapse.

In dendritic spines, the protein synthesis and degradation is modulated by several mechanisms includ-ing RNA interference. Indeed also some miRNAs accumulated at the dendrites in synaptic activity de-pendent manner and correlates with dendritic growth.

Despite several evidence that different mRNAs and miRNAs accumulate depending on synaptic activa-tion, technical limits prevented the uncoupling and identification of dendritic RNA pools from all other cellular RNAs. Up to now the only method is the purification of synaptosomes, that are constituted by both post and pre-synaptic material.

To overcome this limitation, I developed engineered HIV-based Virus–Like Particles (VLPs) to specifical-ly incorporate dendritic RNA species. In particular, I induced the formation of VLPs in dendrites by add-ing a specific localization signal to GAG protein. Moreover, In order to enrich VLPs of miRNAs, I fused a miRNAs binding domains to GAG.

Preliminary results indicated that engineered VLPs form with the expected size and shape and target the dendrites in neuroblastoma cells and in primary hippocampal neurons.

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INTRODUCTION

1. Synaptic Plasticity.

1.1 Hebbian Theory.

In a classical point of view, memory and learning are two theoretical concepts which help us to fix the observation that learning revise the behaviour. In this sense, learning could be defined as the capabili-ty to acquire new information, while memory as the capabilicapabili-ty to maintain information in a state that can be recalled at a later time. In this context, the study of memory is represented by the research of cellular and molecular modifications in the brain (in particular, in the synaptic connections) which take place during learning and memory formation.

The synaptic plasticity is defined as short and long-term changes of synaptic transmission efficiency (also called synaptic strength). The current paradigm in neuroscience gives to synaptic plasticity the role of molecular and cellular mechanism at the base of all cognitive processes. In other words, all the kind of information transfer in the brain occurs at synapses and they are due to synaptic plasticity phenomena.

In order to study and understand the synaptic strength changes, a simple model was provided by Don-ald Hebb, in his book The Organization of Behaviour (1):

When an axon of cell A is near enough to excite a cell B and repeatedly or persistently takes part in fir-ing it, some growth process or metabolic change takes place in one or both cells such that A 's efficien-cy, as one of the cells firing B, is increased.

The Hebb’s principle for the synaptic plasticity states that a synapses is enhanced if there is a temporal correspondence between the activity of the pre-synaptic and post-synaptic terminals. If a neuron A evokes a firing, and it is followed by the firing of the neuron B, then the synapses between A and B is strengthened, because it transmits a significant information. In short words, the synapses which work a lot are enhanced and the synapses which work little are weakened.

Once the Hebb’s principle was assumed as the working hypothesis, it was necessary to find experi-mental models to validate it and to study the molecular mechanisms of the synaptic plasticity. The two most important adopted model are the reflection of the brachial retreat in Aplysia Californica and the phenomena of Long Term Potentiation (LTP) and Long Tem Depression (LTD) in the mammalian hippo-campus. Aplysia Californica is sea snail which is able to retire the gill in response to a little shock. Be-cause of this stereotyped response, it has been an important model to study molecular mechanisms of memory formation (2).

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3 1.2. LTP in hippocampus.

The Hebb’s principle for the synaptic plasticity is an heuristic approach which is valid also for the neu-ral network of high complexity as the mammalian brain. In order to find the phenotypic evidence of this principle, the organ par excellence is the hippocampus, a part of the brain which is implicated in mechanisms of information’s consolidation from short-term memory to long-term memory. It is also involved in spatial memory that enables navigation. The hippocampus belongs to the limbic system and it is part of a structure called hippocampal formation, located under the cerebral

cor-tex (allocortical) and, in primates, in the medial temporal lobe (3). These properties, in addition with the fact that different neuronal cell types are neatly organized into layers in the hippocampus and this represent the reason why it has been widely studied. It contains two main interlocking parts:

the hippocampus proper (also called Ammon's horn) and the dentate gyrus (figure 1) . It elaborates the information from entorhinal cortex and sends them back to the cortex and to the subiculum. The input from the entorhinal cortex occurs through a nerve fiber called perforant pathway, this path con-tains two branches:

- The direct path which projects directly from entorhinal cortex to the CA1 area of the hippocampus. - The indirect path which goes from entorhinal cortex to the granule cells of the dentate gyrus, from granule cells to the giant pyramidal neurons of the CA3 area of hippocampus through the mossy fibers and finally, from CA3 to CA1 through the Shaffer collaterals. All these synapses are excitatory and the principal neurotransmitter is the Glutamate.

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4 Figure 1. Schematic representation of the human hippocampal histology and circuitry.

(Image courtesy of Moseret al., (2011) “The multi-laned hippocampus” Nature Neuroscience; 14, 407–408).

The synaptic plasticity phenomena in the hippocampus are homo-synaptic, namely, they depend on molecular mechanisms inside the synapse itself. They can occur at the pre-synaptic or post-synaptic level. Different forms of synaptic plasticity are subdivided in two groups depending on the time dura-tion:

- Short Term Plasticity (STP) which lasts from milliseconds to minutes. - Long Term Plasticity (LTP) which lasts from hours to years.

At the molecular level, STP is a pre-synaptic phenomena, it is protein synthesis independent and it is due to the homeostasis of calcium in the pre-synaptic terminals (to the residual calcium). On the con-trary, LTP is protein synthesis dependent and can occur at the pre-synaptic or at the post-synaptic level (4).

Experimental evidences of LTP phenomena have been found in all the synapses of the indirect path of the hippocampus, while the molecular mechanisms are different in the different areas:

- In the synapses between the mossy fibers and the CA3 the LTP occurs at the pre-synaptic level. -In the synapses between the CA3 and CA1 (Shaffer collaterals) the enhancements occur thanks to the opening of N-methyl-D-aspartate receptors (NMDARs) present in the post-synaptic terminal mem-branes and to the consequent calcium influx, which lead to the activation of several calcium depend-ent pathways in the post-synaptic neurons. Ultimately, the activation of these pathways leads to an increase of the density of AMPA receptors on the dendritic spines and to the growth of the spines themselves.

NMDARs are particular ionotropic glutamate receptors which can be distinguished from others iono-tropic glutamate receptors (like AMPA receptors) for the presence of a double ligand gaiting, one for the glutamate and one for the magnesium. The Magnesium gaiting is released in a voltage dependent way (figure 2): the membrane depolarization in the post synaptic terminal leads to the release of the blocking pore ion, while, the binding of glutamate and of a second cofactor (glycine or D-serine) leads to the ligand-dependent opening of the channel. The channel opening brings Ca++ influx, which acti-vates several calcium dependent pathways . NMDARs represent a sort of AND logic operator and their opening indicates that the pre-synaptic neuron is firing and that the pre-synaptic activity is not isolat-ed. It indicates that the synapses is carrying information and needs to be enhanced (5).

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5 Figure 2. Schematic representation of NMDA receptor. The opening leads to an increase of different ions membrane permeability. The figure also shows binding sites for different agonists and antago-nists.

(Paoletti et al (2007). “ NMDA receptor subunits: function and pharmacology”. Curr Opin Pharmacol. 7 (1): 39–47)

1.3. Molecular mechanism of LTP.

The Shaffer collaterals NMDA mediated Long Term Potentiation shows very interesting molecular fea-tures. Due to the double control of the NMDAR opening, these synapses have properties of coopera-tiveness, associativity and synaptic specificity.

LTP is a very complex mechanism which could be divided in two steps: the induction, that is protein synthesis independent, and the consolidations, which is a protein synthesis dependent mechanism.

LTP can be experimentally induced by a protocol of high frequency stimulation called tetanic stimula-tion.

The mechanism can be explained with the model of Synaptic tag and capture (STC) formulated by Frey & Morris in 1997 and revised by Redondo & Morris in 2010 (6-7). The synaptic tag is a set of molecular modifications which occur at a synapse and indicate the induction of synaptic plasticity. According to STC theory, the tetanic stimulation induces LTP through the following steps:

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6 - The formation of a synaptic tag in the activated dendrites.

- The synthesis of particular RNAs and proteins by the neuron called Plasticity Related Products (PRPs) , and the distribution of them in the whole cell.

- The PRPs are captured by the synaptic tag.

- The PRPs perform locally their activity leading to LTP. PRPs are RNAs witch are specifically brought to the activated dendrites and expressed. The products work in networks leading to the LTP consolida-tion.

The STC model suggests that there is a whole set of regulatory components which locally takes place at the synaptic level. Therefore, the current paradigm in neurobiology attributes to these molecular mechanisms a central role in the cognitive functions.

One of the most important locally regulatory mechanisms is represented by Calcium/calmodulin-dependent protein kinase type II alpha chain (CAMKIIα). CAMKIIα is a kinase activated by the calmodu-lin in a calcium-dependent way. The peculiarity of this protein is that it is able to stay in an activated state also after its activation (8). CAMKIIα is an holoenzyme with 12 subunits. The opening of NMADR and the subsequent calcium influx bring to the activation of the calmodulin. As shown in figure 3, the couple calmodulin-Ca++ is able to bind the CAMKIIα and to lead to auto-phosphorylation. After the subunits phosphorylation, the enzyme is able to phosphorylate itself and to maintain an activated sta-tus. This behaviour is called toggle switch.

Figure 3. Cartoon that explain the mechanism of CAMKIIα activation.

(Image courtesy of Lisman et al., (1985) “ A mechanism for memory storage insensitive to molecular turnover: a bistable autophos-phorylating kinase”. Proceedings of the National Academy of Sciences, 82(9), 3055{3057 )

According to STC model, CAMKIIα is a Synaptic Tag, in fact it is necessary and sufficient for the induc-tion of LTP. It has several targets through which it promotes the cytoskeleton remodelling, the spine growth, the increase of AMPA channels on the dendritic terminal and the activation of the local

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pro-7 tein synthesis. In fact, local protein synthesis and degradation plays a fundamental role in the consoli-dation of LTP.

1.4. mRNAs localization in neurons.

Neurons are highly polarized cells, and they present strong differences in local proteome in the axonal, somatic and dendritic compartments. These differences are due to:

- The selective transport of proteins from the soma, where most of the protein syntheses processes take place, to the target compartments. It is the case of most of the axonal proteins and of some PRP. - The selective movement of specific mRNAs from the somatic cytoplasm to the target compartments. It is the case of the most of the PRP. This process requires signals in the mRNAs and molecular machin-eries for the specific transport and the protein syntheses, since ribosomes are present in the dendrites.

The selective mRNAs transport and the local protein synthesis, associated with local molecular mecha-nisms of protein and RNAs degradation (ubiquitin-proteasome system and RNA interference (RNAi)) allows an higher degree of control and a faster regulation of local proteome. Therefore, it allows an higher sensitivity and responsiveness of the system.

The signals for the specific mRNAs transport are represented by cis-activating elements called zip-codes, usually located in the 3’UTR of the mRNA molecules. These elements are very different both in sequence and in secondary structure, for this reason they are very difficult to be computationally pre-dicted. The zip-codes are constituted by complex secondary structures and they can present loop ele-ments called RNA-folds. The secondary structure is recognised by particular proteins which act as adaptors between RNAs and molecular motors as Dyneins and Kinesins. Each mRNAs can present more than one zip-code, therefore, different combinations of elements define an unique localization pattern for each RNA species.

The differential mRNAs transport from the nucleus to the target compartment occurs through 1000S ribonucleoproteic complexes called RNA granules (9). These granules are about 30nm in diameter and they are constituted by zip-coded RNAs and by more than 40 proteins with different functions:

- Transport: they form scaffolds that interact with the molecular motors which transport the RNA granules along the microtubules.

-Stabilization: the interactions between mRNAs and some RNA-binding proteins avoid the RNA degra-dation.

-Post-transcriptional regulation: some RNAs-binding proteins can mediate the initiation of the transla-tion of the target mRNAs (for example CPEB in the active states) or can maintain it in a translatransla-tional silent state (RISC complex proteins).

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8 RNA granules contain also stalled ribosomes and other factors implicated in the translational regula-tion.

The RNA granules formation starts in the nucleus, where the transcribed RNAs, after the maturation, start to complex with trans-activated protein factors as the Exon Junction Complex and other RNA-binding proteins which provide the first signal for the localization. Subsequently, these aggregates are exported through the nuclear pore in the cytoplasm where other protein factors as Staufen, FMRP and Zip-Code Binding Proteins (ZBP1 and ZBP2) aggregate around the growing granules. Finally other fac-tors are enrolled in these ribonucleoproteic complexes to give rise to mature granules which get in contact with the molecular motors through protein as Staufen and are brought to the target compart-ment.

RNA granules are very heterogeneous entities both for the RNA and protein content; furthermore, in the cells there are other types of ribonucleoproteic complexes which contain mRNAs as the P-bodies (processing bodies), the Stress granules and Silencing foci.

Several hundreds of mRNAs, which represent about the 3-4% of the cellular transcriptome are en-riched in the dendrites of hippocampal and cortical neurons. Most of these mRNAs, which encode for the structural component of the post synaptic density, are transported in a constitutive way (figure 4).

The specific transport of other mRNAs in dendrites increases in activity dependent way (10).

Figure 4. mRNA enrichment in dendritic compartment. the mRNAs enriched in the dendrites encode for structural and receptor component of the spines.

(Image courtesy of Cajigas et al. (2012) “The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging”. Neuron, 74(3), 453{466 )

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9 1.5. Regulation of protein synthesis and degradation in dendritic spines.

In addition with the dendritic mRNA transport control, in neurons there are several molecular machin-eries which regulate the local proteome through the modulation of mRNA stability and degradation and the protein translation and degradation. These molecular mechanisms can be divided in five groups:

1) Control of translation RNA granules transport: the Fragile X Mental Retardation Protein (FMRP) is a widespread protein in the mammalian brain. It is transported in specific RNA granules, and, during the transport, it takes part in the inhibition of translation of mRNA contained in granules. Its function is mediated by a small non coding RNA called BC1 which acts pairing the target mRNAs. Among them there are important PRP as Arc, CAMKIIα, Map2b. Furthermore, FMRP is able to control the global translation in granules through a direct interaction with ribosomes; in fact, FMRP in a phosphorylated state is able to bind ribosomes in the granule inducing stall. The NMDAR pathway is able to activate the PP2A phosphatase, which is able to dephosphorylate FMRP mediating its detachment from ribo-somes. (11)

2) Another level of control of protein syntheses in response to synaptic activity is represented by RNA interference (RNAi).

3) Non-sense mediated decay (NMD): During the first steps of RNA granules formation, some proteins called Exon Junction Complex (EJC) bind a particular sequence of some mRNAs near exon-exon junc-tions. After the transport, during the mRNA translation, the UPF1 factor is enrolled and interacts with the termination signal of the mRNAs bound to ribosomes, furthermore the factors UPF2 and UPF3 in-teract with the EJC. The complex UPF1, UPF2 and UPF3 leads to the phosphorylation of UPF1 and in this state UPF1 interacts with the initiation factor eIF3 preventing further translation rounds. Due to this control system, after the synaptic activation, there is only a burst of translation of the EJC binding mRNAs. One of these mRNAs is Arc, an immediate early gene that plays fundamental roles in synaptic plasticity. (12)

4) Control of Initiation of translation: Chemical and physical mechanisms which induce synaptic plastic-ity (tetanic stimulus or BDNF administration) are correlated with an increase of the global protein syn-thesis at the local level and with a decrease of the phosphorylation of the alpha subunit of Eukaryotic Initiation Factor 2 (eIF2α). In eukaryotic cells, eIF2 is a G-protein necessary for the initiation of transla-tion, the alpha subunit can be deactivated and sequestrated through phosphorylation performed by several kinases (4 at least). eIF2α can be activated by dephosphorylation performed by phosphatases controlled by several complex pathways connected with synaptic activity. (13) (14)

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10 Another example is represented by Eukaryotic initiation factor 4 F (eiF4F). eiF4F is the translation initi-ation factor which binds the mRNA 5’ cap. Normally, eiF4F is deactivated by interaction with eiF4 bind-ing proteins. These proteins can be separated from eiF4F by phosphorylation performed by several kinases which activity is synaptic plasticity dependent. One of these kinases is S6K which belong to the mTOR pathway (15).

5) Control of local protein degradation through the Ubiquitin-proteasome system: following the activa-tion of NMDAR it is observed a selective recruitment of proteasomes in activated dendritic spines. The prolonged persistence of proteasomes in the dendritic terminals, also after the inducing stimulus, leads to a remodelling of the local proteome. (16)

1.6. Roles of microRNAs in the synaptic plasticity.

In addition with the repression of translation performed by FMRP, there is a second mechanism of pro-tein syntheses control represented by the RNA-Induced Silencing Complex (RISC). The role of mi-croRNAs in dendrites is poorly understood, basically due to the fact that it is technically difficult to un-couple dendritic and somatic RNAs and because of miRNAs high instability. However, several evidences indicate specific roles of RNAi in the synaptic plasticity phenomena.

The central nervous system products an huge amount of miRNAs, most of which have a role that has not be investigated yet. At the neuronal level there is a colocalization between RISC complex proteins and ribonucleoproteic aggregates (RNA granules and P-bodies), most of which contain FMRP. Pivotal studies were performed exploiting the interference of the global miRNAs syntheses or avoiding the function of specific miRNAs by the injection of antisense oligonucleotides. For example, the brain spe-cific miR-132 positively correlates with dendritic growth (17), while the miR-134 is negatively correlat-ed with the dendritic spine size, since it acts by inhibiting the translation of Limk1 that is a target gene of BDNF. Indeed, in response to synaptic activation, the miR-134 is degraded in a local specific way and the Limk1 mRNA is locally translated in a protein that plays a role in microtubule remodelling leading to the dendritic growth (structural synaptic plasticity). (18).

RNAi and the activity dependent protein degradation proteasome mediated can be gathered in a common mechanism. The first evidence was found in Drosophila, investigating on a RNA Helicase of the RISC complex called Armitage (19). As a result of cholinergic activation, Armitage is brought to the dendrites by the RNA granules, and it is degraded by proteasome, hence the protein translation in-creases.

A second study was performed on the mammalian homologous of Armitage, MOV10 (20). MOV10 has a dendritic localization, it is present in the form of puncta constituted by RNA granules and P-bodies. Activating the dendrites with BDNF or tetanic stimulus, the NMDA opening leads to the degradation of

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11 MOV10 mediated by proteasome and to an increase of levels of some synaptic activity related pro-teins, such as Limk1, CAMKIIα and Lypha1 (Figure 5).

Figure 5. The degradation of MOV10 leads to a selective increase of the protein translation. A) and B) protein profiles of free ribosomes (M) respect to polyribosomes (P) obtained by TRAP-seq of control neurons (A) or of MOV10 Knock down neurons; note that, in the second case there is a decrease of free ribosomes and an increase of polyribosomes. C) Relative increases of the proteins MOV 10, CAMKIIα and Lypha1 in control and MOV10 knock down.

(Image courtesy of Banerjeeet al. (2009). “A coordinated local translational control point at the synapse involving relief from silenc-ing and MOV10 degradation”. Neuron, 64(6), 871{884.)

These two evidence suggest that there is an evolutionarily preserved mechanism that controls the translation of the PRP products in synaptic activity dependent way, through miRNAs: some plasticity related products are transcribed and brought to dendrites, where they remain in a sort of priming state, thanks to the paring with miRNAs. As consequence of synaptic activity, the RNAi comes down and these mRNAs are free to be translated and to perform their activity in the explication of synaptic plasticity.

Other studies leads to the discover of few dozen of miRNAs specifically located in dendrites and corre-lated with synaptic activity (21). Currently known miRNAs are shown in figure 6.

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12 Figure 6. Table containing the current known specifically located miRNAs with their targets and roles in synaptic plasticity.

(Image courtesy of Ye et al. (2016) “Role of MicroRNA in Governing Synaptic Plasticity” Neural Plasticity Volume 2016 (2016), Article ID 4959523).

These kind of studies were done knowing the take in account miRNAs before the experiment. Nowadays, there is not an assay that allows to isolate and identify the dendritic miRNA pool (miRNome) since it is technically impossible to uncouple dendritic RNAs from somatic RNAs and because miRNAs present a low stability.

An attempt of dendritic miRNAs screening was done by using Synaptosomes (21).

A synaptosome is an isolated synaptic terminal from a neuron. Synaptosomes are obtained by mild

homogenization of nervous tissue under isotonic conditions and subsequent fractionation using density gradient centrifugation. Liquid shear detaches the nerve terminals from the axon and plasma membrane surrounding the nerve terminal particle reseals. Synaptosomes contain numerous small clear synaptic vesicles, sometimes larger dense-core vesicles and frequently one or more small

mitochondria. They carry the morphological features and most of the chemical properties of the original nerve terminal. Synaptosomes isolated from mammalian brain often retain a piece of the attached postsynaptic membrane, facing the active zone.

In this work, the authors extracted synaptosomes from the forebrain of p15 mice. As control they used the forebrain total RNA and they hybridized the synaptosomes RNA and the total RNA with micro-array that contained probes for all known mature miRNAs. The results are shown in figure 7.

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13 Figure 7. List of miRNAs that displayed an at least twofold change in expression between

synaptosomes and forebrain.

(Image courtesy of Schratt et al. (2009) “A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis” Nature Cell Biology 11, 705 – 716).

However, synaptosomes are not representative of dendritic spines, in fact they are constituted by both the presynaptic and the post synaptic terminals, furthermore the outer surface of synaptosomes represents the inner surface of dendrites, therefore, some dendritic materials is lost during the synaptosomes isolation.

2. HIV-1

The Human Immunodeficiency Virus (HIV) is a lentivirus (a subgroup of retrovirus) that causes HIV infection and over time Acquired Immunodeficiency Syndrome (AIDS) (22). AIDS is a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers. HIV infects vital cells in the human immune system such as helper T cells

(specifically CD4+ T cells), macrophages and dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including apoptosis of abortively infected T cells, apoptosis of uninfected bystander cells,direct viral killing of infected cells and killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells (23). When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections. Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is more

virulent, more infective and is the cause of the majority of HIV infections globally. Because of its relatively poor capacity for transmission, HIV-2 is largely confined to West Africa.For this reasons, the most studies is HIV-1.

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14 2.1 HIV-1 structure and genome

HIV is different in structure from other retroviruses (figure 8). It is roughly spherical with a diameter of about 120 nm. It is composed of two copies of positive single-stranded RNA that codes for the virus's nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein p24, encoded by the HIV GAG gene. (24)The single-stranded RNA is tightly bound to nucleocapsid proteins, p7, and en-zymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease

and integrase. A matrix composed of the viral protein p17, encoded by GAG gene, surrounds the cap-sid ensuring the integrity of the virion particle.

This is, in turn, surrounded by the viral envelope, that is composed of the lipid bilayer taken from the membrane of a human cell when the newly formed virus particle buds from the cell. The viral envelope contains proteins from the host cell and relatively few copies of the HIV envelope protein, which con-sists of a cap made of three molecules known as glycoprotein 120 (gp120) and a stem consisting of three gp41 molecules which anchor the structure into the viral envelope. The envelope protein, en-coded by the HIV env gene, allows the virus to attach to target cells and fuse the viral envelope with the target cell membrane releasing the viral contents into the cell and initiating the infectious cycle (25). As the sole viral protein on the surface of the virus, the envelope protein is a major target for HIV vaccine efforts.

Figure 8. Cartoon represented mature HIV virial particle’s structure.

(Image courtesy of Douek et al (2009). "Emerging Concepts in the Immunopathogenesis of AIDS". Annu. Rev. Med. 60: 471–84)

The RNA genome consists of at least seven structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, and INS), and nine genes (GAG, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat env and rev), encoding 19 proteins. Three of these genes, GAG, pol, and env, contain information needed build a new virus particles: The GAG gene (group-specific antigen) encodes for the virial core protein p24, p17, p9, p7. pol (Polymerase) encodes the fundamental enzymes reverse transcriptase, integrase and protease. While, env (Envelope) encodes the envelope proteins gp120 and

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15 gp41. GAG and pol are transcribed in a single transcript which are translated in a 180 kDa protein that is cleaved by the virial protease. Env gives rise to a single protein which is cut in the two envelope protein by proteolysis. (24)

2.2 HIV life cycle

The HIV life cycle is showed in figure 9. The HIV virion enters macrophages and CD4+T cells by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell. The first step in fusion involves the high-affinity attachment of the CD4 binding domains of gp120 to CD4. Once gp120 is bound with the CD4 protein, the envelope complex undergoes a structural change, exposing the chemokine receptor binding domains of gp120 and allowing them to interact with the target chemokine receptor. (26) This allows for a more stable two-pronged attachment, which allows the N-terminal fusion peptide gp41 to penetrate the cell membrane. Repeat sequences in gp41, HR1, and HR2 then interact, causing the collapse of the extracellular portion of gp41 into a hairpin. This loop structure brings the virus and cell membranes close together, allowing fusion of the membranes and subsequent entry of the viral capsid. After HIV has bound to the target cell, the HIV RNA and various

enzymes, including reverse transcriptase, integrase, ribonuclease, and protease, are injected into the cell. (27)

The capsid is transported to the nuclear pore through microtubules and during the transport, the

reverse transcriptase liberates the single-stranded (+)RNA genome from the attached viral proteins and

copies it into a complementary DNA (cDNA) molecule. The reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that creates a sense DNA from the antisense cDNA. Together, the cDNA and its complement form a double-stranded viral DNA that is transported into the cell nucleus protected by a protein complex named Pre-Integration Complex (PIC). The integration of the viral DNA into the host cell's genome is carried out by another viral enzyme called integrase. This integrated viral DNA may then lie dormant, in the latent stage of HIV infection. To actively produce the virus, certain cellular

transcription factors need to be present, the most important of which is NF-κB, which is upregulated when T-cells become activated.(28)

During viral replication, the integrated DNA provirus is transcribed into RNA, some of which then un-dergo RNA splicing to produce mature mRNAs. These mRNAs are exported from the nucleus into the

cytoplasm, where they are translated into the regulatory proteins Tat (which enhances new virus pro-duction) and Rev. As the newly produced Rev protein accumulates in the nucleus, it binds to length, unspliced copies of virus RNAs and allows them to leave the nucleus. (29) Some of these full-length RNAs function as new copies of the virus genome, while others function as mRNAs that are

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16 translated to produce the structural proteins GAG and Env. GAG proteins bind to viral RNA genomes to package them into new virus particles. (30) During the viral RNA packaging, the nascent virions encap-sidate host RNA, about the 50% of the RNA mass in the mature virus is represented by cellular RNAs. (31)

The final step of the viral cycle, the assembly and budding of new HIV-1 virions, begins at the plasma membrane of the host cell. The Env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by furin resulting in the two HIV envelope gly-coproteins, gp41 and gp120. (32) These proteins are transported to the plasma membrane of the host cell where gp41 anchors gp120 to the membrane. The GAG (p55) and GAG-Pol (p160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell. The budded virion is still immature as the GAG poly-proteins still need to be cleaved into the actual matrix, capsid and nucleocapsid poly-proteins. This cleavage is mediated by the packaged viral protease. The various structural components then assemble to pro-duce a mature HIV virion. Only mature virions are then able to infect another cell. (33)

Figure 9. Simplified representation of fundamental steps of HIV-1 life cycle.

(Image courtesy of Douek et al (2009). "Emerging Concepts in the Immunopathogenesis of AIDS". Annu. Rev. Med. 60: 471–84)

2.3. Capsid assembly and RNA encapsidation.

The full-length HIV-1 genomic RNA (FL RNA) is a capped and polyadenylated Pol II transcript that is selectively incorporated into the viral particles as a noncovalent dimer. (31) RNA packaging into virus

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17 particles is dependent upon specific interaction between FL RNA and the nucleocapsid protein (NC) domain of the GAG precursor. Selection of the HIV-1 genomic RNA involves the so-called Psi region located immediately upstream of the GAG start codon and folded into four stem-loops important for genome packaging (SL1 to SL4)(34). In particular, SL1 contains the dimerization initiation site (DIS), a GC-rich loop that mediates in vitro RNA dimerization through kissing-complex formation, presumably a prerequisite for virion packaging of FL RNA. (35) While both SL2 and SL3 bind HIV-1 NC with high affinity, only SL3 seems capable of independently directing the packaging of heterologous RNAs, pointing at a particular contribution of SL3 in packaging. (36) Additional cis-acting sequences have also been shown to contribute to FL RNA packaging. Some of these elements are located in the first 50nt of the GAG gene, including SL4 and are present only in the FL RNA molecule, whereas others are located upstream of the splice-donor site (SD1), and are consequently present in all HIV-1 RNAs. These upstream sequences include TAR (trans-acting responsive element), poly(A) and U5-PBS hairpins that are involved in FL RNA packaging (37). Although selective encapsidation of FL RNA is largely attributed to Psi, presence of spliced viral RNAs has been found in infectious wild-type (wt) HIV particles and in mutant particles with altered levels of FL RNA due to GAG or Psi mutations, but the determinants and mechanism involved in spliced-RNA encapsidation remain undefined. The FL-HIV-1 transcript

undergoes complex alternative splicing that produces >46 spliced RNAs (38) divided in to two classes: fully spliced mRNAs (2.4 kb) encoding Tat, Rev and Nef, and singly spliced mRNAs (4 kb) encoding Vpu, Vpr, Vif and Env. The fully spliced RNAs, also called the early transcripts, follow the classical route of host mRNAs nuclear export. In contrast, singly spliced HIV-1 mRNA and FL RNA are intron-containing transcripts that would normally be restricted from leaving the nucleus, but are exported via the CRM1-dependent protein export pathway due to the HIV-1 Rev protein, which serves as an adaptor between the Rev-responsive element (RRE) present in the env gene and CRM1(39). Thus, the export pathway might also contribute to the selective packaging of FL RNA. Currently, the packaging efficiency and selectivity of the two groups of spliced RNAs is not well characterized, and one cannot discard the possibility that these spliced viral mRNAs are randomly packaged, together with cellular RNAs. Indeed, retroviruses package significant amounts of cellular RNA (about 50% of the RNA mass in virions) (40).

3. Virus-Like Particles.

In many enveloped-virus, multiple copy of structural antigen (capsid or pericapsid) are able to spontaneously self-assembly in vicinity of plasmatic membranes and budding to give rise at particles which mimic the virions called Virus-Like Particles (VLP). (41) VLPs are biological entities similar to virus, but unable to cause infections since they don’t contain any kind of viral genome. Typically, VLPs contain multiple copies of viral antigens in authentic conformation, so they are immunogenic and

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18 stables, for these properties they are wildly used in medical research and engineered for vaccine

development, cancer treatment, viral assembly studies and in drug delivery. Furthermore, thanks to their physical and chemical properties VLPs provide incredibly regular scaffolds for building at the nanoscale, therefore they are starting to be used in nanotechnology (42).

3.1 Medical application of Virus-Like Particles.

VLPs, mimicking the organization and conformation of authentic native viruses but lacking the viral genome, have been used as immunogenic molecules in several recombinant vaccines in the last few years and have been even used as a therapeutic vaccine to induce the production of specific

antibodies against endogenous molecules with a preponderant role in chronic diseases. Some VLP-based vaccines have been licensed and commercialized (FDA-approved vaccines for Hepatitis B and human papillomavirus are the most well-known examples) and a large number of VLP-based vaccine candidates have also been undergoing clinical evaluation. Therefore, VLPs have provided delivery systems that combine good safety profiles with strong immunogenicity and constituted a safe alternative to inactivated or attenuated vaccines (43) .

In the 1960s, some empty viral particles without nucleic acid were identified as the capsid protein of hepatic B virus (44). This finding was considered as the first recorded instance of the natural existence of VLPs. Subsequently, it was discovered that hepatic B virus VLPs can induce the host immune

responses to eliminate the invasion of the authentic hepatic virus (45). This phenomenon was a clue to understand the relationship between the VLPs and the host immune system. In the 1980s, the antigenicity and immunogenicity mechanism of HBsAg VLPs were interpreted. Hence, VLP-based vaccines gained further attention from researchers.

VLPs structurally resemble the virus from which they were derived and, because of their antigenic similarity to authentic virions and because they possess physical characteristics that are highly immunostimulatory, can serve as effective stand-alone vaccines or vaccine platforms. Most VLPs are between 20–100nm in diameter, a size that allows for free entry into the lymphatic vessels and passive drainage to the subcapsular region of lymph nodes and optimal uptake by professional antigen

presenting cells (APC). The high density of the epitopes on its surface can be recognized and

presented to the immune system by APC, thus stimulating humoral and cellular immunity effectively through similar pathways as the original pathogens do . In some cases, trafficking of VLPs to B cell follicles is facilitated through specific interactions between VLPs and complement components or natural IgM antibodies. VLPs also possess a geometry that contributes to their remarkable ability to activate B cells. Epitopes presented on the multivalent, highly repetitive, and often rigid structures of VLPs can extensively crosslink B cell receptors, leading to strong stimulation of B cells and the induction

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19 of robust and long-lasting antibody responses. These signals can even override B cell tolerance

mechanisms, allowing the induction of potent antibody responses against self-antigens. Thus, these two features, size and geometry, are critical for the ability of VLPs to potently activate B cells and elicit strong and long-lasting antibody responses, and are essential to their utility as vaccines (46) .

Classically, vaccines could be divided in two main group: inactivated vaccines, based on the

administration of purified antigens of pathogens and attenuated vaccines which consist in the injection of living pathogen deleted of some virulence factors. The first group of vaccines is safe, but not so immunogenic and the second group is not safe, since the pathogen sometimes can reacquire the virulence.

As a novel type of vaccine, VLPs offer a solution to the above problem, primarily because of its

biological properties. First, VLPs have a high level of safety without concerns of biosafety since no viral genetic components are introduced during its production. Second, VLPs present conformational epitopes, which are arranged repeatedly on the surface. With such an arrangement, VLPs are more similar to the native virus; thus, VLPs can easily induce strong B cell responses in the absence of adjuvants (47). Third, VLP vaccine can rapidly cope with epidemic viral diseases because of its short time required for proceeding from design to expression. For example, the development and

preparation of VLP vaccines was only 8 weeks after the outbreak of influenza, but more than 5 months for attenuated vaccines (48). Finally, VLPs, as pathogen-associated molecular patterns (PAMPs), can be recognized by pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) of host cells and captured by antigen-presenting cells (e.g., dendritic cells) (49), then can be taken up and processed via the MHC class I pathway (cross-presentation) for activation of CD8+ T cells, which are essential for the clearance of intracellular pathogens such as viruses. In addition, VLPs are inherent in a suitable size and can also be taken up by dendritic cells (DCs) as exogenous antigens for processing and presentation by MHC class II and for directly promoting DC maturation and migration, essential for stimulation of the innate immune response, whose stimulate immunity patterns are similar as in the original virus (figure 10). In this way, VLPs may have the advantages over the cognate live viruses for immune activation because some viruses that replicate in DCs are known to block activation and maturation of the cell through expression of particular viral proteins, while some VLPs which can resemble infectious viruses and retain their receptor binding regions are able to be taken up by antigen-presenting cells for class I presentation systems (43) .

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20 Figure 10. Virus-like particles (VLPs) mimic the overall structure of virus particles.

VLP are recognized readily by the immune system, and present viral antigens in a similar pathway to authentic conformation inducing strong immune responses.

(Image courtesy of Yan ET AL, (2015) ‚‘The application of virus-like particles as vaccines and biological vehicles’’Applied Microbiology and Biotechnology, 24, pp 10415–10432)

Since the 1970s, about 40 infectious diseases have been newly discovered. Vaccination remains an effective tool to reduce this threat, and yet, the conventional cell culture often fails to produce sufficient vaccine dose. Thanks to their properties and the low time and costs of production, as alternative to cell-culture based vaccine, VLPs are considered as a high- priority vaccine strategy against emerging viruses. (50)

VLPs has also been used as delivery vehicles. Ideal biological vectors should have the following biological characteristics: biocompatibility, solubility and uptake efficiency, with targeted delivery and high drug loading. As a nanoscale material, VLPs have high potential in drug delivery. VLPs fit the aforementioned demands in a certain degree among plenty of studies. First, VLPs are easy to be produced in large-scale quantities using the existing expression systems either as enveloped or non-enveloped VLPs. (51) Second, VLPs are capable of targeting the corresponding cell transport with its surface ligands by modification on the gene level (gene insertion) or the protein level (chemical coupling). (52) Third, VLPs have good carrying capacity because of its large surface area and numerous amino acid residues on the surface. (53) Fourth, VLPs are self-assembled by viral structure proteins

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21 under proper conditions which looks more like a protein cage with a large cavity space that can

encapsulate numerous biological molecules, and as a result, expanded these molecules’ applications.(54) Finally, VLPs have thermodynamic stability because of its dodecahedral or icosahedral structure.(43)

To sum, VLPs have emerged as multifunctional platform systems for the development of bio-derived nanomaterials and have good potential for application in drug delivery, genetic therapy, and other fields.

The greatest adverse effect of chemotherapy in cancer treatment is toxicity to normal tissues, which severely limits the therapeutic effects of anticancer drugs. Anticancer drugs are tethered via the amino acid residues on the surface of VLPs, particularly the icosahedral VLPs, such as HBV,

bacteriophage MS2, bacteriophage Qβ, and some dodecahedral VLPs, such as adenovirus (Ad) VLPs (55) with thermodynamic stability. Through mild chemical coupling reagents, anticancer drugs such as adriamycin and aleomycin can be loaded onto the surface of the aforementioned VLPs with hydrazone bonds created by amino acid residues. In addition, anticancer drug molecules can also be

encapsulated into VLPs by the reversible process of self-assembly with changes of the external conditions, such as specific ionic concentration (56). Moreover, VLPs have a proper particle size, good distribution, and biocompatibility as well as ligands on its surface for invasion into special cells. These ligands bind with receptors on the cell surface to help different VLPs to specifically deliver the drugs to various target cells mimicking the native virus. Common ligands are RGD motif (57), transferrin (58), and so forth. At the same time, the anticancer drug bioavailability can be improved, that is, the improvement of the ability of the targeted transport and accumulation in target cells. Therefore, VLPs can be acceptable as effective biological vectors for carrying drugs.

3.2 Virus-Like Particles based on HIV-1 P55 GAG.

The human immunodeficiency virus type I (HIV-1) GAG gene encodes the highly conserved structural polyprotein P55 GAG, which directs the viral assembly process (Figure 11). The GAG protein comprises the major structural proteins p17 matrix (MA), p24 capsid (CA), p7 nucleocapsid (NC), and p6 linker (LI) protein in addition to small spacer peptides p2 and p1, which are released from the GAG precursor by the HI viral protease during or shortly after budding of immature virions from the host cell. The unprocessed GAG precursor itself, in complete absence of any other viral proteins and viral RNA, is sufficient to catalyse the formation and release of non-infectious virus-like particles when expressed in insect, yeast, and mammalian cells. These VLPs are called HIV-1 P55 GAG VLPs or, more simply GAG VLPs and they have similar diameters to wild-type viruses. (59)

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22 Despite the mechanisms of particles assembling has been studying for several years, the kinetics of this process, both in wild type HIV and GAG VLPs, is not completely understood. The uncleaved GAG is a multifunctional polyprotein, containing distinct functional domains that essentially contribute to the HIV assembly process. (60) The membrane binding region of GAG is located in distinct domains within the p17(MA) protein. The N-terminal glycine residue within p17, which serves as the site of myristic acid attachment, has been shown to be crucial for virus assembly and binding of the GAG precursor to the inner leaflet of the plasma membrane. In addition, efficient membrane incorporation of GAG is supported by electrostatic interactions between basic residues within the N-terminus of p17(MA) (aa17–31) and negatively charged phospholipids of the cell membrane. (61) Besides p17(MA), the p24(CA) domain of HIV GAG includes properties which are essential for correct VLP formation.

Mutational analysis within p24(CA) have defined a dimerization domain within the C-terminal third of p24, which has been shown to be essential for GAG multimerization and virion assembly (62) . The p7(NC) domain of GAG is a highly basic region whose role is to encapsulate and protect viral RNA. Two copies of RNA are included in each virion and two zinc finger motifs within the conserved interaction domain of p7 have been mapped to mediate attachment to RNA (63) . Although p7 binds single-stranded RNA relatively non-specifically, it displays a strong preference for viral RNAs containing a Psi-site over cellular RNA. When the GAG gene is expressed in appropriate eukaryotic cells, the GAG polyprotein is synthesized on cytosolic ribosomes and then targeted to the plasma membrane, where it acquires a lipid envelope and buds in form of virus-like particles (Figure 11B and C). During the

cytosolic transport, the GAG precursor seems to form assembly intermediates, recruits single-stranded RNA and then aggregates at the inner face of the plasma membrane as dense patches (Figure 11D), which can be visualized by electron microscopy. The assembled GAG protein complex induces

membrane curvature, leading to the formation of a bud. During budding, viral Env glycoproteins as well as producer cell derived proteins appear to be selectively incorporated into the nascent particles. GAG protein plaques progress to form protruding buds which are released from the plasma membrane to form virus-like particles (Figure 11E) (64) .

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23 Figure 11. Schematic representation of the HIV GAG polyprotein and particle formation. (A) The HIV Pr55GAG polyprotein consisting of the p17 matrix (MA), the p24 capsid (CA), the p7 (NC)- and the p6 linker protein (LI) includes all information to (B) target to the inner leaflet of the membrane of the producer cell and (C) bud in form of enveloped virus-like particles (VLP). (D and E) Electron micrographs of VLPs budding from HEK 293T cells transfected with a plasmid encoding GAG CP: cytoplasm; EC: extra cellular.

(Image courtesy of Deml et al. (2005) “ Recombinant HIV-1 Pr55GAG virus-like particles: potent stimulators of innate and acquired immune responses” Molecular Immunology 42; 259–277)

In HIV virus the RNA is essential for the particles assembly.In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant GAG proteins, apparently by acting as ‘‘scaffolding’’ in the particle. Through RT-PCR and microarray and RNA-seq was demonstrate that, in virus-like particles, the viral genome deputed ‘’space’’ in the capsid, is occupied by other, cell-derived RNA molecules.

Furthermore, the analyses of data reveals that the majority of encapsidated transcripts are evidently represented in the VLPs in proportion to their steady-state expression level in the cells. (65)

The role of RNAs in the GAG VLPs assembling kinetic is not completely understood, however, the current model is reported in figure 12. GAG mRNA is translated in the cytoplasm and the p55 protein takes contact with membrane. Both p17(MA) and p7(NC) have positive charges which are stabilized through the contact with the plasma membrane. In presence of RNA the p7(NC) preferably interacts with it instead of the membrane; these interactions lead to a conformational change of p55 (figure 12 A). In this conformation single p55 molecules are able to assembly in the vicinity of the membrane

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24 inducing membrane curvature and leading to the formation of a bud. Protein plaques progress to form protruding buds which are released from the plasma membrane to form virus-like particles (66) .

Figure 12. Schematic representation of GAG VLP assembly model.

(Image courtesy of Reine et al. (2011) “ Diverse interactions of retroviral GAG proteins with RNAs.” Trends in Biochemical Sciences 36(7):373-80)

4 RNA interference.

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of small ribonucleic acid (RNA) mole-cules, microRNA (miRNA) and small interfering RNA (siRNA), are central to RNA interference (66) . miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, whereas siRNAs derive from longer regions of double-stranded RNA. The human genome may encode over 1000 miRNAs, which are abundant in many mammalian cell types and appear to target about 60% of the genes of humans and other mammals. siRNAs are abundant in plants. miRNAs are small non-coding RNA molecules (containing about 22 nucleotides) which explicate their function through base-pairing with complementary sequences within the 3’UTR of mRNA molecules. (67) As a result, the tar-geted mRNA molecules are silenced, by one or more of the following processes:

-Cleavage of the mRNA strand into two pieces

-Destabilization of the mRNA through shortening of its poly(A) tail

-Less efficient translation of the mRNA into proteins by ribosomes (67)

miRNAs biogenesis is showed in figure 13. miRNAs derive from autonomous appropriated miRNA genes or from introns in other genes; the RNA is spliced, 5’capped and 3’polyadenilated to give rise to a stem loop containing molecule called pri-miRNA. A single pri-miRNA may contain from one to six miRNA precursors. These hairpin loop structures are composed of about 70 nucleotides each. Each

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25 hairpin is flanked by sequences necessary for efficient processing. (68) The double-stranded RNA struc-ture of the hairpins in a pri-miRNA is recognized by the nuclear protein DiGeorge Syndrome Critical Region 8 (DGCR8). DGCR8 associates with the enzyme Drosha, a protein that cuts RNA, to form the Mi-croprocessor complex. In this complex, DGCR8 orients the catalytic RNase III domain of Drosha to lib-erate hairpins from pri-miRNAs by cleaving RNA about eleven nucleotides from the hairpin base (one helical dsRNA turn into the stem) (69). The product resulting has a two-nucleotide overhang at its 3' end; it has 3' hydroxyl and 5' phosphate groups. It is often termed as a pre-miRNA (precursor-miRNA). Sequence motifs downstream of the pre-miRNA that are important for efficient processing have been identified. Pre-miRNA hairpins are exported from the nucleus in a process involving the nucleocyto-plasmic shuttle Exportin-5. This protein recognizes a two-nucleotide overhang left by Drosha at the 3' end of the pre-miRNA hairpin. Exportin-5-mediated transport to the cytoplasm is energy-dependent, using GTP bound to the Ran protein (70).

In the cytoplasm, the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer. This endoribonucle-ase interacts with 5' and 3' ends of the hairpin and cuts away the loop joining the 3' and 5' arms, yield-ing an imperfect miRNA:miRNA duplex about 22 nucleotides in length. Overall hairpin length and loop size influence the efficiency of Dicer processing (71). Although either strand of the duplex may poten-tially act as a functional miRNA, only one strand is usually incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact. The mechanism of loading in RISC complex is not really understood, but it seems that the strand with the less stable 5'end is selected by the protein Argonaute and integrated into RISC. RISC is the complex which perform the RNAi. Mem-bers of the Argonaute (Ago) protein family are central to RISC function. Argonautes are needed for miRNA-induced silencing and contain two conserved RNA binding domains: a PAZ domain that can bind the single stranded 3' end of the mature miRNA and a PIWI domain that structurally resembles

ribonuclease-H and functions to interact with the 5' end of the guide strand. They bind the mature miRNA and orient it for interaction with a target mRNA. Some Argonautes, for example human Ago2, cleave target transcripts directly; Argonautes may also recruit additional proteins to achieve transla-tional repression.(72) The human genome encodes eight Argonaute proteins divided by sequence simi-larities into two families: AGO (with four members present in all mammalian cells and called

E1F2C/hAgo in humans), and PIWI (found in the germ line and hematopoietic stem cells. Additional RISC components include TRBP [human immunodeficiency virus (HIV) transactivating response RNA (TAR) binding protein], PACT (protein activator of the interferon-induced protein kinase), the SMN complex, fragile X mental retardation protein (FMRP), Tudor staphylococcal nuclease-domain-containing protein (Tudor-SN), the DNA helicaseMOV10, and the RNA recognition motif containing protein TNRC6B (73).

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26 Once the single strand miRNA is loaded, the RISC complex is able to scan several cytoplasmic mRNAs until it find a complementary one. In case of siRNA, the pairing is perfect and the Ago cut the target mRNA in two strands which will be degraded by cellular RNAase. Otherwise, the miRNA pair the tar-gets in an imperfect way, the RISC complex performs an inhibition of target translation (74).

4.1 TRBP

The multidomain human TAR element binding protein (TRBP) is a component of the Dicer complex that has been implicated in the processing of pre-miRNAs. TRBP was initially discovered as a protein that associates with the HIV TAR RNA (75) and was later demonstrated to interact with and regulate interferon-induced protein kinase R (PKR), a kinase involved in the cellular response to viral infection (76,77). In the context of miRNA biogenesis, TRBP associates with Dicer, the RNase III enzyme that catalyzes the removal of the apical loop of pre-miRNAs (78) and processes small interfering RNAs. TRBP has been shown to enhance dicing and augment Dicer stability (79), although the molecular

mechanisms for these activities have not been elucidated. Downstream of Dicer, TRBP is also a component of the RISC-loading complex (RLC), the protein assembly responsible for transferring the miRNA/miRNA* duplex from Dicer to RISC (80). TRBP contains three double-stranded RNA-binding domains (dsRBDs), but only the first two dsRBDs interact with dsRNA. The third dsRBD is believed to mediate protein/protein interactions and to bind other proteins in the Dicer complex. The first two dsRBDs are called TRBP-D1 and TRBP-D2 and they have the same shape and dimensions (figure 14). The region of TRBP that binds immature miRNAs comprises these two independent double-stranded RNA binding domains connected by a 60-residue flexible linker. It has been demonstrated that these region interact with both the pre-miRNAs and the dulplex miRNA-miRNA* with affinities in the low micromolar range (81).

Figure 14. cartoon representation of the 3D structures of TRBP-D1 (A) and TRBP-D2 (B) showing the three regions in canonical dsRBDs that are implicated in dsRNA binding.

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27

(Image courtesy of Matthieu et al. (2013) “ The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains.” Nucleic Acids Research. 41(7): 4241–4252)

5. Aim of the thesis.

In the central nervous system, RNA localization plays a pivotal role since the local translation of mRNAs at dendrites can be triggered by synaptic activation and is thought to induce plastic changes that occur at synapses triggered by learning and memory.

In dendritic spines, the protein synthesis and degradation are modulated by several mechanisms including RNA interference. Indeed also some miRNAs accumulated at the dendrites in synaptic activity dependent manner and correlates with dendritic growth.

Nowadays, no one assay allows to isolate and identify the dendritic miRNA pool (miRNome) since it is technically impossible to uncouple the dendritic RNAs from the somatic RNAs and because miRNAs have a low stability. The only available method is the purification of synaptosomes, that are

constituted by both post and pre-synaptic material.

To overcome this limitation, the aim of my thesis is to develop a tool that specifically catch RNA species in dendrites. To this aim I engineered HIV-based virus–like particles (VLPs) that form directly into dendrites due to a dendrite localization signal. Moreover, these modified particles specifically collect miRNAs due to the presence of a specialized binding domain.

5.1 VLPs engineering in order to increase the microRNAs encapsidation.

Whit the aim to enhance the amount of encapsidated miRNAs in VLPs, the GAG protein was fused with the double-stranded RNA binding domains region containing TRBP-D1 and TRBP-D2. The chimeric protein was generated by modifying the plasmid pGAG-EGFP that encode for chimeric fluorescent well assembled Virus like particles (82). The new plasmid was used to transfect HEK 293T cells and to produce engineered VLPs. The integrity and the production efficiency of GAG-TRBP VLPs was checked through WB, fluorescent and electron microscopy analyses. GAG-EGFP VLPs represented the control.

5.2 Engineering of VLPs in order to drive their dendritic expression.

For the purpose of express the GAG protein specifically in dendrites, forcing the VLPs to assembly and bud from the dendritic spines, the PSD-95 Zip code was added in the 3’UTR of chimeric VLP genes. PSD-95 (postsynaptic density protein 95) is a member of the membrane-associated guanylate kinase

(MAGUK) family. it is located in dendritic spines where it is recruited into the NMDA receptor and

potassium channel clusters. PSD-95 interact at postsynaptic sites to form a multimeric scaffold for the

clustering of receptors, ion channels, and associated signaling proteins. Its direct and indirect binding partners include neuroligin, NMDA receptors, AMPA receptors, and potassium channels. It plays an

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28 important role in synaptic plasticity and the stabilization of synaptic changes during long-term

potentiation (83) .

the Zip codes of PSD-95 is a very strong signal, furthermore it has been well studied and defined. It consist in 247 G-quadruplex structure forming base pairs located in 3’UTR of PSD-95 mRNA (84). see figure 15. The engineered zip code contained VLPs have been tested in neuroblastoma cell lines through WB, immunoistochemisty and imaging analyses. Finally, they have been tested in primary hippocampal neurons cultures.

Figure 15. Representation of PSD-95 3’UTR. The circled G-quadruplex structure represent the zip code.

(Image courtesy of Moine et al. (2011) ‘‘G-quadruplex RNA structure as a signal for neurite mRNA targeting‘‘. EMBO Rep. 12(7):697-704)

MATERIALS AND METHODS

1.1 Cloning strategy and plasmids construction.

The plasmid pGAG-EGFP (cat#11468 by M. Resh, obtained through the NIH AIDS reagent program), showed in figure 17, has been used for subsequent cloning strategies. The plasmid pGAG-EGFP encodes for the fusion protein GAG-EGFP. The promoter for the expression of the chimera is the CMV, an ubiquitous promoter which induces a high expression of the protein in several cell types.

The Dendritic Localization Signal (DLS) of PSD-95 is a 247pb sequence, contained in the pEX-A2 Plasmid, already present in the lab. The cloning strategy to insert the DLS in the 3’UTR of the coding sequence of GAG-EGFP or GAG was based on the PCR amplification of the DLS using primers with tails, showed in figure 18. The PCR products were purified using the kit Wizard® and Promega® SV Gel and

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29 restriction enzymes, run on agarose gel and purified. Finally, the DLS containing fragments were ligated downstream the EGFP gene and downstream the GAG gene, respectively (figure 19). The ligation products were transformed into XL10 gold bacterial cells and colonies have been processed to purify the DNA by miniprep. Purified plasmids were checked through digestions and sequencing.

Plasmids pGAG-TRBP and pGAG-TRBP-STOP-DLS, showed in figure 20, were already generated in the lab. The plasmids were checked through RE digestions and the results are showed in figure 21.

Figure 17. Representation of plasmid pGAG-EGFP. The plasmid was obtained by M. Resh through the NIH AIDS reagent program.

Figure 18. Primers used for cloning. The primers couple FW BSRG1-STOP-DLS and REV NOT1-DLS was used to clone GAG-EGFP-STOP-DLS, while, the primers couple FW NOT1-STOP-DLS and REV NOT1-DLS was used to clone GAG-STOP-DLS. The two forward plasmids were designed to insert a STOP sequence (TAA) in frame with EGFP and GAG genes respectively.

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30 Figure 19. Obtained Plasmids. Schematic representation of the final plasmids: A)GAG-EGFP-STOP-DLS B)GAG-STOP-DLS.

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31 Figure 20. Schematic representation of the plasmids: A) GAG-TRBP-STOP. B)GAG-TRBP-STOP-DLS.

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32 Figure 21. Plasmids digestion. (A) pGAG-EGFP checking digestions: Lane 1. digested with BamH1 and Not1. Expected bands 741 bp + 5533 bp. Lane 2. digested with BamH1 and Nde1. Expected bands 1967 bp + 4307 bp. (B) p GAG-EGFP-STOP-DLS and pGAG-STOP-DLS checking digestions. Lane 1. pGAG-EGFP digested with Not1 and BamH1. Expected bands 741 bp + 5533bp. Lane 2-3-4 pGAG-EGFP-STOP-DLS digested with Not1 and BamH1. Expected bands 986bp+5533bp. Lane 5. pGAG-EGFP digested by Age1 and BamH1. Expected bands 618bp + 5656 bp. Lane 6. pGAG-STOP-DLS digested by BamH1 and Age1. Expected bands 863bp + 5656bp. (C) pGAG-TRBP-STOP checking digestions: Lane 1.digested with Not1. Expected band 6132bp. Lane 2. Digested with Sal1. Expected band 6132 bp. Lane 3. Digested with Not1 and Sal1. Expected bands 2125 bp + 4007 bp. (D) pGAG-TRBP-STOP-DLS checking digestions: Lane 1. Supercoild Plasmid. Lane 2. Digested with Sal1. Expected band 6377 bp. Lane 3. Digested with BamH1. Expected bands 5764bp + 631 bp. Lane 4. Digested with Not1. Expected band linearized 6377 bp). Lane 5. Digested with NCO1 (bands 1904 bp + 1858 bp + 1567 bp + 703 bp + 345 bp, last one is

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33 undetectable).

1.2 Cloning strategy and lentiviral vectors production

With the aim of producing VLPs in neurons the constructs GAG-EGFP-STOP-DLS and GAG-EGFP where insert in the Lentiviral vector pLenti-CaMKIIa-eNpHR 3.0-EYFP (cat #26970), showed in figure 22. The advantages of this vector are the expression of EYFP under the control of the neural specific promoter CaMK2α and the ability of highly transduce neurons. The cloning strategy to replace EYFP with GAG-EGFP or GAG-GAG-EGFP-STOP-DLS was based on amplification of the 2 inserts using primers with tails, showed in figure 22. The PCR products were purified using the kit Wizard® and Promega® SV Gel and PCR Clean-Up System. Both the purified PCR products and the lentiviral vector were cut with the restriction enzymes, run on agarose gel and purified. Finally, the inserts were ligated downstream the CaMK2α promoter (figure 24). The ligation products were transformed into XL10 gold bacterial cells and colonies have been processed to purify the DNA by miniprep. Purified plasmids were checked through digestions (figure 25) and sequencing.

The Lentiviral vectors were packaged using HEK-293T cells with PEI reagent. The first day 5*10^6 cells were plated in a p100 dish; the day after cells were cotransfected by 20 µg of the transfer vectors (pLenti GAG-EGFP or pLenti GAG-EGFP-STOP-DLS), 15 µg of the packaging vector pPAX2 and with 5 µg of the VSV-G plasmid per each p100. Supernatants were collected 48h and 72h post transfection, centrifuged at 1200rpm to remove cell debris and filtered by 0.45nm filter. The purified lentiviral vectors were quantified through the kit INNOTEST® HIV antigen mAb (Fujirebio) obtaining the concentrations of 165,3 pg/µl of pLenti GAG-EGFP and 162,5 pg/µl of pLenti GAG-EGFP-STOP-DLS.