Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5

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Thesis

Reference

Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5

LASCANO MAILLARD, Maria Josefina

Abstract

The cellular factor TRIM5α performs a dual role in the innate immunity. First, TRIM5α has an intrinsic ability to induce the AP-1 and NFκB pathways and contributes to the establishment of the LPS-mediated antiviral state. Second, it functions as a restriction factor, blocking early stages of retroviral infection in a capsid-dependent manner. The connections between these two functions of TRIM5α are debated. We investigated the conservation, in TRIM5 orthologues, of the ability to activate the innate immune pathways and analyzed the signification of this function in the context of TRIM5- mediated HIV-1 restriction. We took the advantage that there are seven TRIM5 orthologues in the mouse, with variable abilities to activate the innate immune signaling, to determine the contribution of this signal activator function to the restriction process. [...]

LASCANO MAILLARD, Maria Josefina. Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5. Thèse de doctorat : Univ.

Genève, 2014, no. Sc. 4667

URN : urn:nbn:ch:unige-381368

DOI : 10.13097/archive-ouverte/unige:38136

Available at:

http://archive-ouverte.unige.ch/unige:38136

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITE DE GENEVE

Département de Génétique et Evolution

Département de Microbiologie et Médecine moléculaire

Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5

THESE

Présentée à la Faculté des Sciences de l’Université de Genève Pour obtenir le grade de Docteur ès Sciences, mention Biologie

par

Maria Josefina Lascano Maillard

de

Bassecourt (JU)

Thèse n°4667

Genève 2014

FACULTE DES SCIENCES

Professeur François Karch FACULTE DE MEDECINE Professeur Jeremy Luban

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Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5.

Par

Maria Josefina Lascano Maillard

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ABSTRACT

The cellular factor TRIM5α performs a dual role in the innate immunity. First, TRIM5α has an intrinsic ability to induce the AP-1 and NFκB pathways and contributes to the establishment of the LPS-mediated antiviral state. Second, it functions as a restriction factor, blocking early stages of retroviral infection in a

capsid-dependent manner.

The connections between these two functions of TRIM5α are debated. We investigated the conservation, in TRIM5 orthologues, of the ability to activate the

innate immune pathways and analyzed the signification of this function in the context of TRIM5- mediated HIV-1 restriction. We took the advantage that

there are seven TRIM5 orthologues in the mouse, with variable abilities to activate the innate immune signaling, to determine the contribution of this signal

activator function to the restriction process.

We found that only the TRIM5 orthologues that could activate the innate immune pathways could restrict HIV-1, when fused to the capsid (CA)-binding

cyclophilin A (Cyp or CypA) domain from the owl monkey TRIM5Cyp.

While human TRIM5α poses a potent blockade to N-MLV and EIAV infection, HIV-1 is much less affected in in vitro experiments using laboratory-adapted strains. Although certain gag-protease variants arising from clinical isolates show

an increased sensitivity to human TRIM5α, the involvement of CA and the regions that influence the recognition of these mutants by the restriction factor

are not completely understood. Here, we showed that three regions of the N- terminal domain of HIV-1 capsid are susceptible to modulate the sensitivity to human TRIM5α: the 4^th and 7^th helices, and the cyclophilin A (CypA)-binding

loop.

Taken together our data show the importance of TRIM5-mediated activation of the innate immune signaling in retroviral restriction and suggests a complex

interplay between CA, CypA and TRIM5α during this process.

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RESUME

Le facteur cellulaire TRIM5α possède une double fonction. Premièrement, TRIM5! montre une capacité intrinsèque d’activer les voies de signalisation

cellulaires AP-1 et NFκB et contribue à l’établissement de l’état antiviral enclenché par une stimulation de lipopolisaccharide (LPS). Deuxièmement, il

fonctionne en tant que facteur de restriction, en bloquant la réplication rétrovirale à un stage précoce de l’infection, de manière dépendante de la capside (CA). Les connections entre ces deux fonctions de TRIM5α font l’objet

de débats. Nous avons analysé la conservation de la fonction activatrice d’immunité innée chez des orthologues de TRIM5α et nous avons tenté de déterminer la signification de cette activité dans le contexte de la restriction rétrovirale. Nous avons exploité différents orthologues murins de TRIM5α qui

montrent différentes habiletés à activer les signaux cellulaires de l’immunité innée pour tenter d’établir l’importance de cette fonction dans le processus de

restriction.

Nous avons trouvé une corrélation entre la capacité d’un orthologue de TRIM5α à activer l’immunité innée et son habileté à bloquer le VIH-1, quand ceux-ci sont fusionnés à un domaine de TRIM5-CyclophilinA (TRIM5Cyp) de l’espèce Aotus

trivirgatus, qui lie la capside. Tandis que le TRIM5α humain bloque très fortement la réplication du gammarétrovirus N-MLV et du lentivirus EIAV, le

VIH-1 est beaucoup moins affecté au cours d’expériences réalisées in vitro et chez des souches adaptées au laboratoire. Bien que certains virus avec des variantes de séquences de gag-protease montrent une plus grande sensibilité au TRIM5α humain, le rôle de CA et l’influence des différentes regions de CA sur cette susceptibilité ne sont pas clairement établis. Nous avons montrés que trois

régions du domaine N-terminal CA sont susceptibles de moduler la reconnaissance par TRIM5α : les hélices 4 et 7, ainsi que la boucle liant la cyclophiline A (CypA). Nos résultats montrent l’importance l’activation de l’immunité innée par TRIM5α dans la restriction rétrovirale et suggèrent une

intéraction entre CA, CypA et TRIM5α durant ce processus.

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Acknowledgments

First of all, I would like to thank my supervisor, Professor Jeremy Luban that allowed me to conduct a thesis in his lab. We spoke very interesting science together and he supported me even when I had crazy ideas that I wanted to test!

Next, I want to thank Professor François Karch, to have accepted to be the co-‐

director of my thesis. Thanks to the two members of the jury, Dr. Angela Ciuffi and Dr. Dominique Garçin, to have kindly accepted my invitation. A special thanks to the latter that, together with Professor Laurent Roux, our former dear department Director, to have welcomed me for their lab meetings and have given me advises for my research. Additionally, Dominique’s laugh is contagious!

I am also very thankful to Professor William Kelley, because of his kindness and his precious help with the English language corrections on my thesis

introduction.

Thanks to my friend and colleague, Hanni Bartels. I will never forget the coffees we drank together, or our Pain quotidien on Fridays!

Thank you to Manel and Anastasia, to the nice lunches spent together and to have adopted me after my dear friend Hanni moved away!

I want to thank Madeleine for her sweetness and for the very nice dinners in her beautiful terrasse! Thanks to Stéphane that coached me in my beginnings and that was of very good support later. It was nice to speak French with him!

Thanks to my other colleagues, Massimo, Alberto, Federico, Christian, Dario, and Jessica. Or should I say (the remaining one now have the capacity to understand anyways): Grazie milla!

I wanted to thank my husband Julien for its support and sweetness during my entire thesis and for being part of my life. He allows me to be a better person each day. He also gave the more beautiful present I could have dream of: mi muchachito Augustin, the sunshine of our home!

Thanks to my mum and my dad, Adriana and José for all the love they gave to my brothers and me, as well as for encouraging us in every moments. We are very lucky to have them! Thanks to my brothers Santiago and Clemente for all the great moments we share together and for always being present if I need support.

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Thank you to all my family for making Augustin so happy each time that he goes to visit.

Thank you to Geneviève and Jean, for their kindness and for the nice walks in the mountains as well as the table game moments spent together.

Thanks to Anne-‐Marie, Nina and Patrick, for having me welcomed in their family, for their sweetness and for all the good moments spent together!

Last but not least, I would like to dedicate this manuscript to my beloved

grandmother Josefina Christe that always believed in me and supported me. She will always be present in my heart and thoughts.

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List of Abbreviations

cDNA Complementary DNA CypA Cyclophilin A

TRIM5Cyp TRIM5-cyclophilin A RF Ring finger domain BB B-box domain CC Coiled-coil domain T12A TRIM12A

T12B TRIM12B T12C TRIM12C T30A TRIM30A T30B TRIM30B T30C TRIM30C T30D TRIM30D

CA Capsid MA Matrix PR Protease gRNA Genomic RNA RSV Rous sarcoma virus

AIDS Aquired Immunodefficiency Syndrom HIV Human Immunodeficiency Virus

HEK293T Human embryonic kidney 293T fibroblasts IRES Internal ribosome entry site

PPT Polypurine tract LTR Long terminal repeats

miRNA MicroRNA

shRNA Short hairpin RNA MLV Murine leukemia virus

EIAV Equine Infectious Anemia Virus

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MSCV Murine stem cell virus ERV Endogenous retrovirus APC Antigen presenting cells DC Dendritic cell

TLR Toll-like receptor NLR Nod-like receptor RLR RIG-I-like recptor

PRR Pattern recognition receptor LPS Lipopolysaccharide

mRNA Messenger RNA PIC Preintegration complex

RTC Reverse transcription complex PBS Phosphate buffered saline PCR Polymerase chain reaction Luc Luciferase

RT Reverse Transcriptase tRNA Transfer RNA

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Chapter 1

INTRODUCTION

1.1.1 Retroviruses

Viral replication requires the transcription and translation machinery, which they themselves lack, from the organisms that they infect.

The common feature among all retroviruses, and also what makes them unique among other viruses, is that they reverse transcribe their RNA genome into DNA that can be inserted into the host genome ¹ (figure 1). For this process, retroviruses encode an RNA-‐dependent DNA polymerase, an RNAseH, and a host-‐encoded transfer RNA (tRNA) that serves as the primer for reverse transcription.

Discovered and first studied as disease-‐causing agents, many decades of research has uncovered the molecular mechanisms governing spread, replication and disease progression caused by these viruses.

Retroviruses can produce fast and slow-‐progression diseases including various types of tumors and immunodeficiency ^2,3.

A principal characteristic of these viruses is that they integrate into the host genome ⁴. This is the reason why, they later became to be used as vectors for gene delivery into cells ^5-‐7.

If the integration event happens in the germline, the retroviral sequences can be spread from one generation to the next, a phenomenon called vertical transmission⁸. This feature of retroviruses is exploited to trace the evolution of genes of the host species, which inherits retroviral sequences in a Mendelian fashion, and used in the study of speciation ^9,10.

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A subset of retroviruses lead to cell transformation and cancer, therefore they are designated as oncoviruses ¹¹. The investigation of the corresponding insertion sites within the genome thus lead to the identification of genes involved in cell growth and tumor promotion ¹². Certain oncoviruses have inherent transforming potential owing to the prior acquisition of host sequences

13This characteristic is useful for the study of genetic regulation of the cell growth ¹⁴.

Figure 1: The retroviral life cycle. The main steps of the retroviral replication cycle are depicted. Blue:

capsid; yellow: nucleocapsid; black bars within the nucleocapsid: RNA genome; orange bars: DNA genome.

Courtesy of Prof. Jeremy Luban (adapted).

1.1.2 The discovery of retroviruses and the reverse-‐transcriptase enzyme

The first retroviruses were discovered at the beginning of the twentieth century as oncogenic agents affecting birds. Ellermann and Bang found that leukosis in poultry was caused by a factor present in ultra-‐filtered cell extracts ¹⁵, that was later called Avian Leukemia virus. A few years later Rous showed that an agent

Expression Assembly

Nuclear transport Budding Maturation Binding

Membrane Fusion

Reverse Transcription

Integration

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present in cell-‐free extracts was the intrinsic cause of the sarcoma formation in the fowl ^5,16. This retrovirus was named after the man who discovered it, the Rous Sarcoma Virus (RSV).

Almost thirty years later, a murine virus was found to be the agent provoking leukemia in mice and thus was called Murine Leukemia Virus (MLV) ¹⁷.

MLV belongs to the gammaretrovirus genus of the retroviridae family ¹⁷.

The concept of an RNA virus converting its genetic material into a DNA form and integrating into the host genome was not yet formulated. The only information scientists had at that moment was that the viral agent did not have a DNA genome but instead was constituted of RNA ⁵.

In the beginning of the 1960s, the molecular biologist Howard Temin worked with the RSV and found that inhibiting the DNA synthesis blocked the viral replication ¹⁸. This led him to propose the provirus hypothesis. That is, the retroviruses have a DNA intermediate in the cells that they infect.

Later, in the year 1970 his team and another virologist involved in the MLV research published separately data showing the presence in RNA tumour virus particles -‐ called virions -‐ of a RNA-‐dependent DNA polymerase activity, by correlating the induced RNA degradation with the decrease of the DNA synthesis.

This enzyme was later called the reverse-‐transcriptase ^13,19,20.

In humans, certain types of acute leukemias were studied and the viral cause of this disease was soon investigated. A type C morphology (that will be defined in the following section) retrovirus, which close relative had been discovered to induce leukemia in the Gibbon ape was pointed out by Robert Gallo to be the cause of the human disease and it was called Human T cell Leukemia Virus (HTLV) ²¹.

The previous findings provided the biochemical and molecular tools that ultimately allowed the subsequent identification of the Human

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Immunodeficiency Virus type 1 (HIV) as the agent causing the Acquired Immunodeficiency Syndrome (AIDS) ²².

In the next section, I will introduce the general retroviral structure.

1.1.3 The general structure of retroviruses

It all starts with the viral RNA. The positive single-‐stranded RNA genome is composed of different regulatory sequences and open reading frames (ORFs) ¹² (figure 2), and has a 5’ cap and a poly A tail.

The regulatory elements are located at the extremities of the viral RNA and consist of repeated (R) sequences, a unique 5’ sequence (U5) containing a cis-‐

acting attachement (att) site, a unique 3’ sequence (U3), the primer binding site (PBS), the psi (packaging signal) element (ψ) and a polypurine tract (PPT) ¹² (figure 2).

The R regions are redundant in sequence and are found after the m7G5’ppp5’Gmp cap, which mimics the eukaryotic mRNA 5’cap. The U5 sequence is immediately downstream of the 5’ R sequence and contains the att sequence that is involved in proviral integration. These regions are followed by the PBS where the specific tRNA primer hybridizes and starts the transcription of the minus-‐strand DNA (-‐sDNA). The next sequence in the RNA genome is the ψ region recapitulating most of the sequences required for viral genome packaging into the viral particles. A major splice donor site, that gives rise to different subgenomic mRNAs, often closely follows this element. Subgenomic RNAs are different mRNA species created when reverse transcription jumps on the template in the 3’ to 5’ orientation. The resulting mRNAs have variable 5’ regions overlapping with the template strands at different levels but the same 3’

sequence. The generation of various mRNAs allows condensing a high amount of information ¹².

The PPT, positioned at the 3’ end of the viral genome, consists of a row of purines Adenine and Guanosine, required for the initiation of the +sDNA transcription.

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Finally, the U3 region preceding the polyA tail contains another att site and in addition a set of cis-‐regulatory sequences essential for viral gene expression.

Given that the synthesis of the viral DNA involves a duplication of the extremities of the RNA templates with a subsequent transfer of the U5 and U3 regions, the two ends in the resulting dsDNA are identical and these are called Long Terminal Repeats (LTRs) ¹².

The provirus is integrated and found in the host genome with the flanking LTRs

5. When the provirus is transcribed, the 5’ U3 region is not taken into account and the synthesis proceeds until the R to U5 boundary. In this way, the resulting viral RNA has the same genomic organization as the template from viral particles.

The viral proteins are encoded by three ORFs, namely the group antigen (gag), the polymerase (pol) and the envelope (env). These genes code for precursos that once cleaved will give rise to more than one protein.

The gag ORF codes for the matrix (MA), the capsid (CA) and the nucleocapsid (NC) ¹².

The pol gene products are the protease (PR), the reverse-‐transcriptase (RT), the integrase (IN) and, in some cases, a dUTPase.

Finally, the precursor synthesized from the env gene is cleaved into the surface envelope protein (SU) and the transmembrane envelope protein (TM) ¹².

Once processed from their precursors the viral proteins form the mature virion, which is able to infect susceptible cells that express the appropriate receptors.

The viral core of a mature viral particle consists in the diploid RNA genome that interacts with the NC, creating a condensation, surrounded by the CA protein complex. The matrix protein that covers this core is surrounded on top by a host-‐

derived lipid bilayer and the included SU and TM proteins ¹²

The viral core contains as well the pol-‐derived proteins that will be used for a novel round of replication, namely the PR, the RT and the IN ¹².

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Figure 2: Schematic view of the proviral genome structure of retroviruses. The retrovirus proviral DNA is composed of untranslated regions that flank the ORFs for gag, pro, pol, env and in some cases that of accessory genes. The flanking LTRs contain U3 and U5 regions, as well as a repeat sequence (R). The 5’

region of the retroviral genome is followed by a PBS and a psi encapsidation signal. Adjacent to the last ORF, the viral RNA contain a PPT. ORF: open-‐reading frame; LTR: log terminal repeats; U3 and U5: unique regions 3 and 5, respectively; att: attachemetn site; PBS: primer binding site; PPT: poly-‐purine tract. Adapted from Fouty and Solodushko, 2011 ²³ .

1.1.4 The reverse-‐transcription process.

Once the retroviral genome enters the cell, the diploid single-‐stranded genome that is still bound to the nucleocapsid (NC) protein, constituting the viral core, starts the process of reverse transcription ^24,25.

For reverse transcription to take place, important elements contained in the viral particles are required. The central component is the reverse transcriptase enzyme, which catalyzes four different reactions: RNA-‐dependent and DNA-‐

dependent DNA polymerization, DNA strand separation via its helicase function and the hydrolysis of the RNA fragments on RNA-‐DNA heteroduplexes ²⁶. The viral core carries additionally a specific collection of transfer RNA (tRNA)

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molecules, different cellular messenger RNAs (mRNAs) from previously infected cells and some ribosomal RNA (5S and 7S) ²⁶.

Reverse transcription starts when the 3’ region of a specific tRNA is used as a primer that anneals with the PBS within the 5’ region of the viral RNA genome (figure 3). DNA synthesis continues until the 5’ extremity of the RNA strain is reached, resulting in a short DNA strand called the minus strand strong stop DNA (–ssDNA) ²⁷.

The next step takes the advantage that the minus-‐strand DNA contains a repeat (R) sequence that is present at both viral genome termini and that was introduced in the newly synthesized DNA molecule by the reverse transcription of the 5’ region of the viral RNA. This confers a complementarity of the –sDNA and the 3’ end of the RNA genome that allows the transfer of the small oligonucleotide to that region, after that the RNAse H function of the RT has degraded the RNA to which the newly synthesized DNA is annealed. This marks the beginning of the elongation of the –sDNA chain, with an accompanying RNA degradation accomplished by RNAse H ²⁷.

During the RNA dependent-‐DNA synthesis, the ppt permit the RNA to escape degradation and this RNA fragment is then used as a primer for the plus-‐strand DNA (+sDNA) polymerization that finally reaches the U5 region of the –sDNA. In the mean time, the –sDNA continues to be polymerized, with a subsequent gradual RNA degradation.

In the following step, the +sDNA synthesis proceeds until the level of the PBS complementarity is formed and the RNA and tRNA primers are degraded. When the tRNA is removed from the +sDNA a complementarity region is exposed and the second strand transfer happens where the plus and minus strands anneal.

The resulting molecule is a circular DNA intermediate ²⁷.

This point of the viral replication cycle can lead to a non-‐productive dead-‐end DNA molecule which contains a single LTR or to a productive DNA form flanked

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by two LTRs, resulting from the strand displacement of the plus and minus strands and resulting in the DNA synthesis towards the PBS and the ppt ²⁷.

Figure 3: The reverse-‐transcription process. The viral RNA is converted to DNA by the reverse-‐

transcriptase enzyme. The first step is the binding of the tRNA primer to the PBS. Subsequent synthesis of the short minus –sDNA and its further annealing with the 3’ LTR of the viral genome will initiate the synthesis of the –sDNA. When the –sDNA reaches the PBS, the +sDNA starts to be synthesized. The resulting molecule is a dsDNA molecule with two flanking LTRs and that can be inserted into the host genome. -‐

sDNA: minus-‐strand DNA; +sDNA: plus strand DNA. From Hu and Hugues, 2012 ²⁸.

1.1.5 The classification of retroviruses

Retroviruses are members of the Retroviridae family. Depending on the morphology of the particles, their structure and their genomic sequences, the Retroviridae family can be divided into seven genera, further regrouped on the basis of their complexity.

The group-‐specific antigen (gag), protease (pro), pol and envelope (env) gene products are encoded by all genera of Retroviridae. Complex retroviruses carry in addition accessory genes with different regulatory functions. The simple

cleavage is detected while RT is actively synthe- sizing DNA; instead, cleavages occur at sites where DNA synthesis pauses (Driscoll et al.

2001; Purohit et al. 2007). Whatever the exact mechanism, RNase H degradation removes the 5⁰end of the viral RNA, exposing the newly synthesized minus-strand DNA (see Fig. 1).

The ends of the viral RNA are direct repeats, called R. These repeats act as a bridge that allows the newly synthesized minus-strand DNA to be transferred to the 3⁰end of the viral RNA. Retro- viruses package two copies of the viral RNA

genome; the first (or minus-strand) transfer can involve the R sequence at the 3⁰ends of either of the two RNAs (Panganiban and Fiore 1988; Hu and Temin 1990b; van Wamel and Berkhout 1998; Yu et al. 1998). After this transfer, minus-strand synthesis can continue along the length of the genome. As DNA synthesis proceeds, so does RNase H degradation. How- ever, there is a purine-rich sequence in the RNA genome, called the polypurine tract, or ppt, that is resistant to RNase H cleavage and serves as the primer for the initiation of the

R U5 pbs gag pol env ppt U3 R

R U5

pbs gag pol env ppt U3 R

pbs gag pol env ppt U3

R U5

R U5 r A

r A

R U5 U3

R U5

pbs gag pol env ppt

LTR LTR

U3 R U5

U3 A B

C

D

E

F

G

Figure 1.Conversion of the single-stranded RNA genome of a retrovirus into double-stranded DNA. (A) The RNA genome of a retrovirus (light blue) with a tRNA primer base paired near the 5⁰end. (B) RT has initiated reverse transcription, generating minus-strand DNA (dark blue), and the RNase H activity of RT has degraded the RNA template (dashed line). (C) Minus-strand transfer has occurred between the R sequences at both ends of the genome (see text), allowing minus-strand DNA synthesis to continue (D), accompanied by RNA degradation. A purine-rich sequence (ppt), adjacent to U3, is resistant to RNase H cleavage and serves as the primer for the synthesis of plus-strand DNA (E). Plus-strand synthesis continues until the first 18 nucleotides of the tRNA are copied, allowing RNase H cleavage to remove the tRNA primer. Most retroviruses remove the entire tRNA; the RNase H of HIV-1 RT leaves the rA from the 3⁰end of the tRNA attached to minus-strand DNA. Removal of the tRNA primer sets the stage for the second ( plus-strand) transfer (F); extension of the plus and minus strands leads to the synthesis of the complete double-stranded linear viral DNA (G).

HIV-1 Reverse Transcription

Cite this article asCold Spring Harb Perspect Med2012;2:a006882 3

www.perspectivesinmedicine.org

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retroviruses consist of the Alpharetroviruses, Betaretroviruses and Gammaretroviruses ¹².

Alpharetroviruses infects a large range of birds. They assemble at the cell membrane and possess a central spherical core (C-‐type morphology). The tRNA they use for the priming of reverse transcription is the one for tryptophan (tRNA^Trp). A typically well-‐studied member of this genius is the Avian Leukosis Virus (ALV) and the previously mentioned RSV ¹².

Members of the Betaretroviruses infect different mammalian species including mice and primates. Morphologically, they can have either an asymmetric round core, either a cylindrical one. They contain a dUTPase gene in frame with the pro gene and they use the tRNA^Lys. The oncovirus Mouse Mammary Tumor Virus (MMTV) is a member of this family ¹².

Gammaretroviruses possess C-‐type virion morphology. They have two ORFs.

The first one encodes the gag, pro and pol gene products; the second one encodes the envelope proteins. The tRNAs used by these retroviruses are mainly the ones for proline or glutamine. Highly documented oncogenic members of this genius include the Murine Leukemia Virus (MLV), Feline Leukemia Virus (FLV) and Gibbon Ape Leukemia Virus (GALV) ¹².

The group of complex retroviruses is composed of Deltaretroviruses, Epsilonretroviruses, Lentiviruses and Spumaviruses. Deltaretroviruses and Epsilonretroviruses have a similar C-‐type virion morphology. The first genius is composed of members encoding two accessory proteins named rex and tax, which are involved in the synthesis and processing of viral RNA. It uses the tRNA^Pro. An example of this group is the oncovirus Human T-‐Lymphotropic Virus 1 (HTLV-‐1) and the closely related HTLV-‐2. The second genius is uses the tRNA for histidine or arginine and codes additionally for three proteins called ORFA, B and C respectively. The function of these accessory proteins are not well understood but in the case of the better-‐studied member Walley Dermal Sarcoma Virus (WDSV), ORFA has been shown to be an orthologue of

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mammalian cyclin c, ORFB activates the PKC and AKT signaling and ORFC has oncolytic properties ²⁹.

The AIDS-‐causing HIV-‐1 belongs to the genius of lentiviruses and is characterized by a conical shape of the core of the mature virion. Members of this group carry this name because of the long asymptomatic phase preceeding the first symptoms ¹². HIV-‐1 expresses six accessory proteins that will be discussed below. These gene products control transcription, gene expression and assembly and counteract restriction factors encoded by the host ¹². The primer used by lentiviruses is the tRNA^Lys3.

In latin Spuma means foam. The members of the Spumaviruses produce vacuolization of cells, hence resulting in a foamy-‐like histological aspect. The human foamy virus is a well-‐studied member of this group. The pol gene products arise from a splice transcript. Unlike other retroviruses, this genius of viruses is characterized by virions that carry high amounts of reverse-‐

transcribed DNA. Accessory proteins shared by the members of this group include a transcriptional transactivator. The primer used is generally tRNA^Lys ¹².

1.1.6 The Acquired Immunodeficiency Syndrome (AIDS)

The AIDS is a severe disease affecting more than 35 millions of people around the world, as published by the UNAIDS report on the global AIDS epidemics 2013

30.

In the early 1980s, young men with typical immunodeficiency symptoms were hospitalized in Los Angeles, New York and California ^31,32.

As mentioned previously, biochemical and genetic tools for studying retroviruses existed in that decade and they were used by Researchers at the Institut Pasteur and in the United States to characterize the virus extracted from CD4+ T cells coming from AIDS patients. Barré-‐Sinoussi and colleagues isolated and described a virus that was able experimentally to infect T lymphocytes

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extracted from cord blood ²² and called it Lymphoadenopathy Associated Virus (LAV).

The team of Robert Gallo, had suspected that the causing agent of AIDS was of retroviral origin and possessed T-‐cell tropism but at that time attributed it to the human tumor retrovirus HTLV-‐I ³³. The virus was later called HTLV-‐III by the same team.

In 1986, the virus was finally named the Human Immunodeficiency Virus (HIV), in reference to the disease it produced ³⁴.

Transmission of HIV-‐1 from one person to another happens during sexual intercourse, injecting with contaminated needles, or by blood transfusion ³⁵. Mother to child transmission during delivery or after breast-‐feeding is another important route of spreading ³⁵.

The first events of HIV-‐1 infection seem to implicate a local spreading within cells residing in the mucosa and in the epithelium, such as dendritic cells (DCs), CD4+ T cells and macrophages ^36-‐38. Primary infected cells subsequently migrate to the lymphoid organs and seed the virus by direct cell-‐to-‐cell contact or by the release of newly produced cell-‐free viruses, which enter new cells ³⁹.

When HIV-‐1 gp120/gp41 glycoproteins interact with the lectin receptor DC-‐

SIGN at the surface of DCs, the virus can be either endocytosed and degraded within lysosomes or by targeting to the proteasome ^40,41. Another route for entry into DCs is mediated by a host-‐derived glycosphingolipid present in the virion envelope that binds to an unknown receptor, with SIGLEC-‐1 being a potential candidate ⁴². This interaction allows the virus to escape degradation and join immunological synapses, from where new target CD4+ T cells can be reached

12,43,44.

During the acute phase of infection, a large fraction of CD4+ T cells are infected and high amounts of virions are synthesized and released from cells ³⁹. As CD8+

T cells fight against the pathogen and high doses of type I interferon (IFN) and cytokines are released, infected individuals commonly experience flu-‐like

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symptoms ^45-‐47. The immune response mediated by cytotoxic T cells and B cells producing antibodies permits to moderately recover the level CD4+ T cells for a few weeks ⁴⁷. At that point, HIV-‐1 already integrated into the host chromosomes and latent reservoirs starts to be established, and infected individuals can have a total absence of HIV-‐1-‐related symptoms for nearly ten years ⁴⁷. Unfortunately, in the meantime, the virus continues to replicate and spread via the various lymphoid organs.

At the terminal stage, the disease causes a high destruction of the CD4+ T cells, which decrease below 200 cells per mm³ of blood, leading to immune suppression and the subsequent unavoidable infection by opportunistic pathogens as Candida albicans and Pneumocystis jirovesii ⁴⁸.

The AIDS pandemic is likely to have originated in central Africa as a result of cross-‐species transmission of a chimpanzee lentivirus to humans. Studies of sequence homology between SIVcpz and HIV-‐1 have shown that the human lentivirus is derived from the simian one ⁴⁹. The second type of HIV, named HIV-‐

2, is less pathogenic and transmissible and thus less frequently leads to AIDS.

Although the two viruses have a similar genome organization, they are derived from different SIV strains ⁴⁹. Whereas HIV-‐1 comes from the SIVcpz, HIV-‐2 arose from a zoonosis with the sootey mangabey monkey, Cercocebus atys. Instead of the Vpu accessory protein, HIV-‐2 possesses Vpx, which counteracts a block to reverse transcription within DCs and macrophages ⁵⁰. Additionally, HIV-‐1 and HIV-‐2 highly diverge from their env sequence. In fact, it was observed that there is already 25% of divergence of the gag, pol and env sequences within the strains of each type, as reviewed by Reeves and Doms ⁵¹.

A combination of nucleoside or non-‐nucleoside reverse-‐transciptases inhibitors and protease inhibitors constitute an aggressive therapy for maintaining the virus load at a low level ^52,53. The highly active antiretroviral therapy (HAART) allowed the life expectancy of individuals to reach nearly normal life spans ⁵⁴. Thanks to these combined anti-‐retroviral therapies and efforts employed in prevention education, new infections have diminished of near 30% compared to

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2001 (UNAIDS report, 2013). However, the pathogen is still far from being eradicated as HIV-‐1 has rarely been totally cleared from an individual ^55,56 and a vaccine is still missing.

The reasons why the search for an effective vaccine has been unsuccessful until now could be in part the inability of the immune system to detect a dormant virus and inherent to the tropism of the virus that targets to destruction the immune cells themselves ⁴⁴. Another important point that could explain the failure of the immune system to detect HIV-‐1 and mount a robust response is that this virus does not productively infect the DCs that are antigen-‐presenting cells (APCs), that prime the immune effectors to kill infected cells. Yet the antigen from these cells is being presented. It seems likely that the APC needs to be activated for the priming of effector T cells to be efficient, an unproductive infection leading to no immune activation will fail to fulfill this prerequisite ⁴⁴.

The innate immunity actors and consequences of their activation will be introduced further below.

1.1.7 The structures of the HIV-‐1 virion and genome

The HIV-‐1 gag orf codes for a precursor polyprotein of 55 kDa in size, called Pr55^gag, which is cleaved within the virion into the MA, the CA, the NC proteins and p6 that is involved in viral budding ⁵⁷.

HIV-‐1 membrane form a spherical particle that has a diameter of approximately 110 nanometers (figure 4). The virion contains a conical-‐shaped CA protein complex that is composed of 216 hexamers and 12 pentamers, linked between them by the C-‐terminal domains of CA ^58,59(figure 4).

The viral particle core is enclosed by a layer of MA proteins, in turn surrounded by a lipid bilayer coming from previous infection events.

The MA protein form hexameric higher-‐order complexes, which encapsulate the viral core. These complexes interact with different virion components and seem

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to be essential for various processes. The well demonstrated bindings include the interaction with envelope bilayer through myristoylated motifs, a process essential for virion assembly at the plasma membrane ⁵⁷. The MA interacts as well with phosphatidiylinositol-‐4,5-‐bisphosphate, leading to the targeting of the myristoyl tails to the plasma membrane and helping the MA to bind to the viral genome ^60,61. Although subject to controversy, MA was reported to bind to the inner domain of gp41, stabilizing the interaction of the envelope into the assembling virion ⁵⁷. Additionally, the Pr55^gag-‐derived protein was shown to interact with the reverse-‐transcription and pre-‐integration complexes (RTC and PIC, respectively), suggesting a role of the MA complex in the early viral life-‐cycle steps.

The CA protein is divided into two structural domains. The N-‐terminal domain stabilizes the structure of the virion and is in the outer layer of the viral core. The C-‐terminal domain faces the inner space containing the genome and contributes to link the hexameric and pentameric rings together. The hexameric lattice of CA interacts with different cellular factors. Its binding to the restriction factor TRIM5 inhibits both reverse-‐transcription and further pre-‐integration steps (see below). As it will be further discussed later, the CA interacts with endogenous Cyclophilin A.

When Cyclophilin A is part of a TRIM5 orthologue protein, HIV-‐1 is bound and strongly restricted ⁶². The restriction mediated by the TRIM5 protein and orthologues will be discussed in more detail in the next sections.

Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5

Thesis

Reference

Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5

Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5

Innate immune signaling and the contribution of different regions of capsid to HIV-1 restriction by TRIM5.

Acknowledgments

Table of contents

List of Abbreviations

Chapter 1