Characterization of a Novel Tetraspanin-Rich, HIV-1-Containing Compartment in Dendritic Cells

(1)

Thesis

Reference

Characterization of a Novel Tetraspanin-Rich, HIV-1-Containing Compartment in Dendritic Cells

GARCIA, Eduardo

Abstract

Dendritic cells (DC) are crucial components of the early events of HIV infection. DC capture and internalize HIV-1 at mucosal surfaces and efficiently transfer, in the absence of viral replication in DC, the virus to CD4+ T cells through infectious synapses (trans-infection pathway). Alternatively, HIV-1 replicates in DC, the resulting DC-T cell viral transfer resulting exclusively from de novo produced viral particles in infected DC (cis-infection pathway). Our results clearly indicate that, for both pathways, HIV-1 is internalized into a novel, non-lysosomial, tetraspanin-rich compartment in DC. Subsequently, the differential recruitment, during the cis-infection pathway, of the AP-3 complex (adaptor protein complex 3), a complex involved in the viral protein Gag targeting to the membranes of late endosomes/MVBs, argues in favour of a potential role of the tetraspanin-rich compartment in HIV-1 production in DC.

GARCIA, Eduardo. Characterization of a Novel Tetraspanin-Rich, HIV-1-Containing Compartment in Dendritic Cells. Thèse de doctorat : Univ. Genève, 2008, no. Sc. 4000

URN : urn:nbn:ch:unige-60000

DOI : 10.13097/archive-ouverte/unige:6000

Available at:

http://archive-ouverte.unige.ch/unige:6000

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

1 / 1

(2)

UNIVERSITE DE GENEVE

Département de biologie moléculaire FACULTE DES SCIENCES

Professeur Robbie Loewith

Département de dermatologie et vénéréologie FACULTE DE MEDECINE et département de microbiologie et médecine moléculaire Professeur Vincent Piguet

_____________________________________________________________________

Characterization of a Novel Tetraspanin-Rich, HIV-1-Containing Compartment in Dendritic Cells

THESE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Eduardo GARCIA de

Meyrin (GE)

Thèse N° 4000

GENEVE

Centre d’Impression Universitaire 2008

(3)

1. RESUME EN FRANCAIS

Avec 33.2 millions de personnes vivant avec le VIH à la fin de l’année 2007 et malgré une prévalence parmi la population adulte stable depuis plusieurs années (www.UNAIDS.org), l’épidémie liée au VIH est loin d’être enrayée. Une interprétation exacte des données à disposition souligne clairement la dureté de l’épidémie ainsi que ses implications sur un plan humain et économique, notamment dans les pays en voie de développement (Piot et al., 2001).

La transmission du VIH-1 intervient principalement via l’incorporation de particules virales présentes dans les fluides biologiques tels que le sang, les sécrétions vaginales, le sperme et le fluide cérébrospinal (Flint et al., 2004). Malgré l’importance de certaines voies de transmission liées à des situations particulières, comme l’échange de seringues entre toxicomanes, la voie principale de transmission du VIH-1 résulte des rapports sexuels non-protégés (Flint et al., 2004).

Au cours de ce processus, les virions doivent traverser les muqueuses (vaginales et/ou rectales) afin de « coloniser» un nouvel hôte. Les propriétés naturellement défensives de l’epithelium des muqueuses expliquent pourquoi l’accès du VIH-1 vers les sous-muqueuse est hautement facilité au niveau de brèches dans la barrière mucosale (Pope and Haase, 2003; Shattock and Moore, 2003). Une fois pénétré, le VIH-1 établit l’infection localement avant qu’elle ne se propage de manière systémique à travers tout l’organisme (Haase, 2005). La conséquence clinique majeure de l’infection par le VIH-1 est la disparition progressive et irréversible des lymphocytes T CD4⁺ entrainant chez les personnes infectées des réponses immunes inefficaces contre des pathogènes usuellement contrôlés (Kuritzkes and Walker, 2007).

La barrière mucosale une fois franchie, le virus se trouve confronté aux sentinelles majeures de la surveillance immune : les cellules dendritiques (DC). En coopération avec d’autres cellules du système immunitaire, les DC contribuent grandement à l’instauration de réponses spécifiques, contrôlées et efficaces contre les microorganismes, allant des parasites aux virus (Banchereau and Steinman, 1998). Présentes dans les tissus périphériques et lymphatiques sous forme immature (iDC), les DC sont capables de surveiller et répondre efficacement à leur environnement. La détection d’un « intrus » entraine son internalisation et dégradation en antigènes destinés à être présentés aux cellules effectrices via des récepteurs histocompatibles, conférant aux DC un statut

« professionnel » de présentation antigénique. Les propriétés de reconnaissance et présentation antigéniques font de la DC l’acteur majeur du lien entre immunité innée et acquise. Lors de l’acquisition d’antigènes, les DC initient leur maturation, un programme développemental

(5)

transformant les iDC hautement endocytiques et faiblement immunogéniques en DC matures (mDC) faiblement endocytiques et hautement immunogéniques. La concomitante migration vers les organes lymphoïdes permet aux mDC d’activer de manière spécifique des lymphocytes immunologiquement naïfs à travers des contacts cellule-cellule appelés synapses immunologiques. Les contacts productifs entrainent ainsi une expansion massive et clonale de cellules lymphoïdes effectrices spécifiques de l’antigène initialement détecté aboutissant à une réponse immunitaire acquise adéquate (Banchereau and Steinman, 1998; Lanzavecchia and Sallusto, 2001b).

La plupart du temps efficaces envers de nombreux pathogènes, les remarquables fonctionnalités des DC semblent être mises à profit par le VIH-1. En effet, les DC dégradent la majorité des virions internalisés dans les 24h (Moris et al., 2004; Turville et al., 2004), aboutissant à la présentation d’antigènes viraux aux lymphocytes T instaurant ainsi une réponse immune apparemment capable à court terme de contrôler l’infection (Flint et al., 2004). Toutefois le statut

« privilégié » des DC en tant qu’APC offre au virus une voie d’accès incontournable vers les lymphocytes T CD4⁺, sa cible de prédilection. Qu’elles soient elles-mêmes infectées ou non, les DC ont été décrites comme étant capables d’augmenter l’infection VIH-1 de cellules T au sein d’une co-culture et ce à des niveaux bien supérieurs à ceux d’une infection de cellules T seules (Cameron et al., 1992; Granelli-Piperno et al., 1998; Pope et al., 1994). La description récente de synapses infectieuses (nommées ainsi à cause de nombreuses similitudes avec la synapse immunologique) entre des DC porteuses du VIH-1 et des cellules T CD4⁺ a clarifié, d’une certaine manière la robuste infection des cellules T médiée par les DC (McDonald et al., 2003;

Piguet and Sattentau, 2004). Des investigations approfondies ont mené à la proposition selon laquelle les DC sont capables de transmettre l’infection VIH-1 lors d’un processus à deux phases (Turville et al., 2004). Au cours des 24 premières heures d’exposition au VIH-1, les DC, de part leur capacité d’endocytose élevée, lient des particules virales de VIH-1 grâce à des récepteurs de surface (tel la lectine de type C, DC-SIGN) et internalisent des virions, la majorité duquel sera dégradée (Moris et al., 2004; Turville et al., 2004). Néanmoins, une faible fraction du virus internalisé échappe à la dégradation et est transféré, en l’absence de réplication virale dans les DC, vers les cellules T à travers une synapse infectieuse DC-T (Arrighi et al., 2004a; Arrighi et al., 2004b; Garcia et al., 2005; Lore et al., 2005; Turville et al., 2004). La seconde phase plus tardive décrit un transfert de particules virales résultant exclusivement de la production de novo de virions du VIH-1 dans les DC (Garcia et al., 2008; Turville et al., 2004).

4

(6)

Plusieurs laboratoires dont le notre, ont démontré le rôle essentiel de la lectine de type C DC- SIGN dans la trans-infection de cellules T CD4⁺ en absence de réplication virale dans les DC (Arrighi et al., 2004b; Geijtenbeek et al., 2000b; Kwon et al., 2002; Sol-Foulon et al., 2002).

Comme partie intégrante de mon travail de thèse, j’ai notamment participé à l’élucidation du rôle de DC-SIGN dans ce processus. En invalidant l’expression de DC-SIGN à la surface des DC et grâce à la microscopie confocale à immunofluorescence, nous avons pu montrer que DC-SIGN, malgré son rôle dans la liaison de particules virales, n’est pas primordial pour l’internalisation du virus mais joue un rôle majeur lors de la formation de la synapse infectieuse (Arrighi et al., 2004a). En effet, la diminution d’expression de DC-SIGN clairement réduit la fréquence des synapses infectieuses entre des DC traitées avec VIH-1 et des cellules T, le virus internalisé ne se relocalisant pas, dans les DC DC-SIGN^-, d’un compartiment intracellulaire à la zone de contact cellules-cellules (Arrighi et al., 2004a).

Bien que l’internalisation du VIH-1 après capture soit cruciale pour le transfert viral vers des cellules T (Arrighi et al., 2004a; Kwon et al., 2002; McDonald et al., 2003), rien n’était connu au sujet de la nature du compartiment, en quelque sorte, protecteur dans lequel se retrouve le VIH-1 dans les DC. Grâce à l’utilisation d’un virus utilisant CXCR4 (non ou faiblement réplicatif dans les DC), nous avons pu caractériser, dans des DC dérivées de monocytes, un nouveau compartiment intracellulaire non-classique, non-lysosomal et enrichi en tetraspanines CD81 et CD9 (Garcia et al., 2005). Faisant usage de la microscopie confocale à immunofluorescence et de la microscopie électronique, nous avons pu montrer que, comme le compartiment classique des endosomes tardifs/MVB, le compartiment, riche en tetraspanines, contenant le VIH-1 est aussi caractérisé par la présence de vésicules intraluminales (Garcia et al., 2005). De manière frappante, nous avons pu aussi démontrer que, après contact avec des cellules T, les DC contenant du virus capturé relocalise ce dernier à l’interface entre la DC et la T (la synapse infectieuse), simultanément avec CD81. Vu que CD81 semble jouer un rôle dans la synapse immunologique, nous avons suggérer que le VIH-1 semble détourner une voie pré-existante de routage de tetraspanines vers la synapse immunologique (Garcia et al., 2005).

Puisque l’infection des DC semble jouer un rôle prépondérant dans le transfert à long terme de particules virales entre DC et cellules T (Lore et al., 2005; Nobile et al., 2005; Smed-Sorensen et al., 2005; Turville et al., 2004), nous avons tenté de décrire l’acheminement précis de particules virales nouvellement synthétisées dans les DC . Après avoir observé qu’un virus utilisant CCR5 se retrouve, après capture, dans le même compartiment enrichi en tetraspanines, nous décrivons

(7)

une distribution intracellulaire dans un compartiment CD81/CD9-positif des particules virales nouvellement synthétisées dans les DC dérivées de monocytes (Garcia et al., 2008). Nous observons aussi, lors de la réplication virale dans les DC, le recrutement différentiel du complexe AP-3 (adaptor protein complex-3), un complexe impliqué dans le ciblage de Gag vers les membranes des endosomes tardifs/MVB (Dong et al., 2007), sur le compartiment enrichi en tetraspanines (Garcia et al., 2008). Conjointement, ces données arguent un rôle potentiel du compartiment dans la production de VIH-1 dans les DC.

Collectivement, les résultats présentés ici contribuent à disséquer les complexes interactions entre DC et VIH-1 et potentiellement aident à la compréhension des phénomènes centraux de la transmission mucosale du VIH-1. Bien que n’étant pas les seules actrices des étapes précoces de l’infection VIH-1, les interactions entre DC et cellules T dans les muqueuses vaginales ou rectales contribuent sans aucun doute à l’essentiel de la dissémination systémique du virus (Haase, 2005). Des investigations plus approfondies mèneront certainement à une connaissance plus juste du court laps de temps à disposition pour contrecarrer l’infection naissante, permettant ainsi le développement de vaccins et de microbicides puissants et efficaces (Haase, 2005; Nikolic et al., 2007).

6

(8)

2. ABSTRACT

With 33.2 million people living with HIV by the end of 2007, and although HIV-1 prevalence among the adult population has remained stable for a number of years (www.UNAIDS.org), the HIV epidemics is far from over. Accurate and honest interpretations of the data at hand clearly underline the harshness and the implications of the pandemic regarding human and economical costs, which in particular burden the efforts made by developing countries (Piot et al., 2001).

HIV-1 transmission occurs mainly through the incorpotation of viral particles in various body fluids such as blood, vaginal secretion, semen and cerebrospinal fluid (Flint et al., 2004).

Regardless of the importance of some in particular situations, namely needle-sharing among drug users, the number one route of HIV-1 transmission worldwide is through unprotected sexual intercourse (Flint et al., 2004). During this process, HIV-1 virions must cross the vaginal or rectal mucosal barrier in order to gain a foothold in the new host. The natural protective properties of mucosal epithelia explains why the access of HIV-1 to the submucosa is greatly facilitated by breaches in the mucosal barrier (Pope and Haase, 2003; Shattock and Moore, 2003). Once in, HIV-1 virions establish the infection locally before it spreads systemically throughout the whole body (Haase, 2005). The major clinical outcome of HIV-1 infection is the progressive but irreversible decline of CD4⁺ T cells in infected individuals and the subsequent, although not immediate, inability to mount appropriate immune responses against otherwise innocuous pathogens (Kuritzkes and Walker, 2007).

Upon crossing the mucosal barrier, HIV-1 viral particle are met with the first line of defense against incoming pathogens, namely dendritic cells (DC). Along with other cells of the immune system, they greatly contribute to a specific, controlled and efficient response to pathogenic microorganisms, ranging from parasites to viruses (Banchereau and Steinman, 1998). Present in peripheral and lymphoid tissues as immature DC (iDC), DC are able to very efficiently scan and properly answer their environment. Detection of a foe leads to its internalization and degradation into antigens destined for presentation to effector cells of the immune system. DC stand therefore at the crossroads between innate and adaptive immunity and it is through the innate recognition of pathogens that DC are able to instigate a proper adaptive response (Guermonprez et al., 2002).

Upon antigen uptake DC go through maturation, a developmental program that turns them from highly endocytic/poorly immunogenic iDC into poorly endocytic/highly immunogenic mature DC (mDC). Concomitant migration to lymph nodes allows mDC to specifically activate

(9)

immunologically naïve T cells through cell-to-cell contacts called immunological synapses.

Productive contacts therefore drive their massive and clonal proliferation, two pre-requisites for an effective adaptive immune response (Banchereau and Steinman, 1998; Lanzavecchia and Sallusto, 2001b).

Regardless of how efficient they might be against many incoming pathogens, DC can not cope with HIV-1. Indeed DC degrade the majority of incoming virions within 24 hrs (Moris et al., 2004; Turville et al., 2004) and are able to present HIV-derived antigens to T cells, therefore initiating in immune response that, at first, seems to control HIV infection (Flint et al., 2004). But the “priviledged” status of DC as APC gives the virus access to CD4⁺ T cells, its favorite target.

Whether themselves infected or not, DC have been described to enhance HIV-1 infection of co- cultured T cells to levels clearly superior than HIV-1 infection of T cells alone, (Cameron et al., 1992; Granelli-Piperno et al., 1998; Pope et al., 1994). The recent description of infectious synapses, thus called because of obvious parallels with the immunological synapse, between an HIV-1-carrying DC and an uninfected CD4⁺ T cell clarified to some extent why DC-mediated infection of T cells is so robust (McDonald et al., 2003; Piguet and Sattentau, 2004). Further investigations led to the proposal that DC are able to transfer HIV-1 infection to T cells in a two- phase process (Turville et al., 2004). Within 24 hrs of exposure to HIV-1, DC, because of their elevated endocytic capacity, bind HIV-1 viral particles through cell-surface receptors (like the C- type lectin DC-SIGN) and internalize virions, the majority of said virions being degraded (Moris et al., 2004; Turville et al., 2004). Nevertheless, a diminutive fraction of captured virions escapes degradation and is transferred, in the absence of viral replication in DC, to T cells through a DC-T cell infectious synapse (Arrighi et al., 2004a; Arrighi et al., 2004b; Garcia et al., 2005; Lore et al., 2005; Turville et al., 2004). The later second phase describes transfer of viral particles resulting exclusively from de novo production of HIV-1 virions in DC (Garcia et al., 2008; Turville et al., 2004).

Previous data from our lab and others demonstrated that the C-type lectin DC-SIGN plays an essential part in DC-mediated HIV-1 trans-infection of CD4⁺ T cells in the absence of viral replication in DC (Arrighi et al., 2004b; Geijtenbeek et al., 2000b; Kwon et al., 2002; Sol-Foulon et al., 2002). As part of my thesis work, I notably took part in the elucidation of the role of DC- SIGN in this process. From our DC-SIGN knockdown and confocal immunofluorescence data, we conclude that DC-SIGN, although responsible for HIV-1 particles binding, does not mediate internalization of virions by itself but plays a role downstream of capture, in infectious synapse

8

(10)

formation. DC-SIGN downregulation clearly reduced the frequency of infectious synapse formation between HIV-1-pulsed DC and autologous CD4⁺ T cells, as internalized captured HIV- 1 virions failed to relocalized from an internal compartment to the cell-to-cell zone of contact in DC-SIGN^- DC (Arrighi et al., 2004a).

Although HIV-1 internalization after capture was shown to be important for viral transfer to T cells (Arrighi et al., 2004a; Kwon et al., 2002; McDonald et al., 2003), nothing was known about the nature of the somehow protective HIV-1-containing compartment in DC. Using a non- replicative (in DC) CXCR4-using-HIV-1 strain, we were able to characterize, in monocyte- derived DC, a novel, non-classical, non-lysosomal intracellular compartment enriched in the tetraspanins CD81 and CD9 (Garcia et al., 2005). Using confocal immunofluorescence and electron microscopy, we could show that just like the classical late endosome/MVB compartment present in DC, the tetraspanin-rich/HIV-1-containing compartment also contains intraluminal vesicles (Garcia et al., 2005). Strikingly, we also highlight that, upon contact with CD4⁺ T cells, HIV-1-pulsed DC relocalized trapped HIV-1 virions to the DC-T cell zone of contact (the infectious synapse) in conjunction with CD81. Because CD81 plays a role in the immunological synapse, we conclude that HIV-1 seems to highjack a pre-existing pathway of tetraspanin sorting to the immunological synapse (Garcia et al., 2005).

Since DC infection plays a crucial role in the long-term transfer of HIV-1 viral particles to CD4⁺ T cells (Lore et al., 2005; Nobile et al., 2005; Smed-Sorensen et al., 2005; Turville et al., 2004), we set out to investigate the precise intracellular trafficking of de novo synthesized HIV-1 particles in DC. After assessing how replicative CCR5-using-HIV-1 is internalized in the same tetraspanin-rich compartment as CXCR4-HIV-1, we describe an intracellular distribution of newly synthesized viral particles in monocyte-derived DC, once again colocalizing to the CD81/CD9-rich compartment (Garcia et al., 2008). The differential recruitment of AP-3, an adaptor protein complex involved in HIV-1 Gag trafficking to late endosomal/MVB membranes (Dong et al., 2005), makes a compelling argument in favor of a role of the tetraspanin-rich compartment in HIV-1 production in DC (Garcia et al., 2008).

Taken together the results presented here further highlight the intricate interactions at play between DC and HIV-1 and might help understand the increasingly complex phenomena occurring during HIV-1 mucosal transmission. Although not the only actors present at the very early steps of HIV-1 infection, DC-T cell interactions at the portal of entry (i.e. the vaginal or

(11)

rectal mucosa) are certainly significant contributors to the systemic spread of HIV-1 infection (Haase, 2005). Further investigations will certainly lead to a better understanding of the small time window at hand to prevent systemic infection, enabling the fine tuning of potent and effective mucosal vaccines and microbicides (Haase, 2005; Nikolic et al., 2007).

10

(12)

3. ABREVIATIONS

AP adaptor protein

APC antigen presenting cell

CLR C-type lectin receptor

CTL cytotoxic T lymphocyte

DC dendritic cell

DC-SIGN DC-specific ICAM-3-grabbing nonintegrin

ESCRT endosomal sorting complex required for transport

GALT gut-associated lymphoid tissue

HIV-1 human immunodeficiency virus

iDC immature dendritic cell

LC Langerhans cell

MDDC monocyte-derived dendritic cell

MHC-I & -II major histocompatibility complex class-I and –II

MVB multivesicular body

myDC myeloid dendritic cell

PAMPS pathogen-associated molecular patterns

pDC plasmacytoid dendritic cell

PRR pathogen-recognition receptor

R5-HIV-1 CCR5-using HIV-1

TEM tetraspanin-rich microdomain

X4-HIV-1 CXCR4-using HIV-1

(13)

4. GLOSSARY

cis-infection pathway A mode of DC-mediated infection of CD4⁺ T cells which requires DC infection and de novo synthesis of progeny viral particles in DC for long-term transmission of HIV-1 infection.

trans-infection pathway A second mode of DC-mediated infection of CD4⁺ T cells which does not require DC to be infected. DC capture and internalize infectious particles, later transferred to target cells, in the absence of viral replication in DC.

HIV-1-pulsed DC DC which, after exposure to HIV-1, capture and internalize virions but are not infected (no viral replication occurs).

‛resting’ CD4⁺ T cells When used in this manuscript, refers to in vivo CD4⁺ T cells which exhibit a resting phenotype but however support HIV-1 replication, unlike in vitro resting CD4⁺ T cells.

infectious synapse Refers to cell-to-cell contacts through which HIV-1 is transferred from an effector cell (i.e. DC) to a target cell (i.e. CD4⁺ T cell), whether the effector cell is infected or not (e.g. HIV-1-pulsed DC). In the literature, when the effector cell is infected, these structures are often referred to as virological synapse. For the sake of clarity, we do not make this distinction in this

manuscript.

12

(14)

5. INTRODUCTION

5a. HIV-1 pathogenesis and AIDS

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of the acquired immunodeficiency syndrome or AIDS (Barre-Sinoussi et al., 1983; Popovic et al., 1984;

Sarngadharan et al., 1984). Together with HIV-2, an immunologically distinct but related virus prevalent in a number of West African countries (Freed and Martin, 2007), HIV-1 is responsible for a tremendous epidemic that continues to spread across the globe with each passing year.

According to the joint United Nations program on HIV/AIDS (UNAIDS), 33.2 million people were living with HIV at the end of 2007. In addition, 2.5 million new infection cases and 2.1 million AIDS-related deaths have been reported for the same period (www.UNAIDS.org).

Although these estimates may seem encouraging when compared with those of 2006 (38.6 million people living with AIDS, 4.1 million new infection cases and 2.8 million AIDS-related deaths), the apparent decline in the number of people living with HIV worldwide sadly result mainly from methodological improvements during survey. A better and more accurate population-based understanding of HIV’s epidemiology, coupled with the extension of relevant surveillance sites in many countries are among the major elements leading to such overall decrease (www.UNAIDS.org). The HIV epidemic remains nevertheless a serious worldwide health problem today and for the years to come. Qualitative interpretations of the data at hand still underline the severity and the implications of the pandemic in terms of human and economical costs, especially for countries under intense developmental efforts (Fig.1) (Piot et al., 2001).

Figure 1. HIV prevalence throughout the world. Although this map represents the situation at the end of 2006, it is still valid for 2007 as HIV prevalence as remained stable. (source: WHO/UNAIDS)

(15)

As discussed later on, HIV-1 cell tropism is determined by the sequential use of CD4 and a co- receptor as cellular receptors during entry into susceptible cells. As a consequence, CD4⁺ T helper lymphocytes, CD4⁺-harboring macrophages and some CD4⁺ dendritic cells (DC) subsets, all important players of the immune system, are the main targets of HIV (Freed and Martin, 2007). The major clinical outcome of HIV-1 infection is the progressive and irreversible decline of immune cell numbers (namely CD4⁺ T helper cells) in infected individuals and the subsequent failure to mount appropriate immune responses towards pathogens mostly harmless in healthy individuals (Fig. 2) (Kuritzkes and Walker, 2007). Progression towards AIDS can be divided into three stages (Flint et al., 2004). During the acute phase, HIV-1 replicates to high levels in activated CD4⁺lymphocytes, producing flu-like symptoms in infected individuals (Kahn and Walker, 1998). Central to the pathogenesis of the virus, HIV-1 infects ‛resting’ memory CD4⁺ T cells in mucosal lymphoid tissues, notably the gut-associated lymphoid tissue (GALT), inducing a severe and irreversible depletion in T cells numbers within these tissues (Brenchley et al., 2004;

Mehandru et al., 2004). In most cases, HIV-1 replication is reduced to low levels within the first few weeks of infection, most likely via the cell-mediated immune response, as the number of circulating activated CD8⁺ T cells (or cytotoxic T lymphocytes (CTL)) increases before apparition of neutralizing antibodies. Seroconversion, from HIV-1-negative to HIV-1-positive, is used by physicians as a clear marker of HIV infection (Gaines et al., 1987; Schmitz et al., 1999).

Momentary control of viral replication leads to total CD4⁺ T cells counts rebounding to almost pre-infection levels (Fig. 2). Following resolution of the first phase, the residual level of viremia, the so-called virological set point, serves as an accurate indicator of disease progression, with high viremia indicating a likely fast progression to AIDS. From a clinical point of view, this stage of the HIV-1 infection seems devoid of symptoms for patients, hence its delusive definition as the asymptomatic phase. Absence of symptoms results from the delicate equilibrium between the ongoing viral infection and its containment by the host immune system. During this period that can last for years in untreated patients, CD4⁺ T cells slowly but steadily disappear (30 to 60 cells/µl of plasma/year) (Mellors et al., 1997) while viral replication continues in lymph nodes, regardless of relatively high counts of activated CD8⁺ T cells (Fig. 2). The deadly combination of the potential direct cytopathic effect of HIV on CD4⁺ T cells and the simultaneous CD8⁺ T cell- mediated killing of infected lymphocytes are the main factors behind the steady drop in CD4⁺ T cells. Furthermore, HIV-1 preferentially infects HIV-1-specific memory CD4⁺ T cells, thus eliminating the very cells that might respond to it (Douek et al., 2002). During the asymptomatic phase, opportunistic infections and malignancies related to AIDS are rare events in HIV-infected individuals as long as CD4⁺ T cells counts remain above 500 cells/µl. AIDS-related symptoms

14

(16)

start appearing below these values but only drastically increase when CD4⁺ T cells counts reach below 200 cells/µl. From then on, the risk of life-threatening complications all caused by otherwise innocuous pathogens such as Pneumocystis jiroveci (formerly, P. carini) pneumonia, systemic fungal infections, cryptococcal meningitis and malignancies related to previous viral infections, drastically increases, leading to further assaults on the immune system and finally death (Kuritzkes and Walker, 2007; Masur et al., 1989).

Asymptomatic phase (persistent state)

Acute Development of AIDS

CD8⁺T cells

CD8⁺ anti‐HIV response CD4⁺T cells

5 10 15 2 4 6 8 10 12

Weeks Years

Time after HIV infection

Relative no. of CD8+cells

10² 10⁷

copies/ml of plasmaCD4+cells (cells/µl) 0 200 400 600 800 1, 000 1, 200

Figure 2. Schematic representation of the course of HIV-1 infection in untreated patients. From (Flint et al., 2004).

A full understanding of the reasons behind the inability of the host immune system to control HIV infection remains elusive. Accumulating evidences however underline that, along with viral immune escape, HIV-1-mediated killing of CD4⁺ T helper cells and the profound architectural modification of lymph nods contribute greatly to this situation (Kuritzkes and Walker, 2007;

Stevenson, 2003). While in theory, virus-specific CD4⁺ T helper cells, central to both humoral and cellular immune responses, assist CD8⁺ T cells in an effective CTL-response that contains chronic viral infections (Day and Walker, 2003; Wherry et al., 2003), loss of the HIV-1-specific CD4⁺ T helper cells population renders CD8⁺ T cells functionally ineffective (Douek et al., 2002;

Flint et al., 2004). Development of potentially effective treatments over the years has led to the emergence of potent combinatory antiretroviral therapies. Since its introduction in the mid-90s, highly active antiretroviral therapy (HAART) significantly and strongly reduced AIDS-related

(17)

mortality and morbidity in developed countries (Egger et al., 2002; Ickovics et al., 2002; Parker et al., 1991). Patients under such regimen exhibit HIV-1 levels around the limit of viral RNA detection (50 copies of HIV RNA per ml of plasma) (Flint et al., 2004). HAART does not however confer viral clearance and HIV-1 replication still occurs below detection levels in specific tissue compartments, such as resting CD4⁺ T cells, macrophages and most likely DC.

Known collectively as cellular reservoirs, these viral sanctuaries allow HIV-1 to resist in the face of highly suppressive therapies and host immune responses (Finzi and Siliciano, 1998; Stevenson, 2003). Among several factors, poor compliance to often complex and barely tolerable regimen is another major contributor to HAART failure (Ickovics et al., 2002). The appearance of multiple drug-resistant forms of HIV-1, due to the inherent poor fidelity of HIV-1 genome replication, further adds a layer of complexity in HIV-1 treatment, making development of even more effective antiretroviral therapies a difficult challenge (Coffin, 1995; Little et al., 2002). Because a successful vaccine has yet to prove safe and effective (Kaiser, 2008), it is of the outmost importance that further studies continue to dissect HIV’s viral life cycle and pathogenic behavior, aiming to discover new potential therapeutic targets. New insights will hopefully lead to efficient, affordable means to both prevent and eradicate HIV-1 infection.

5b. The phylogeny of HIV-1

HIV-1 is an integral member of the Retroviridae family and as such is an enveloped RNA virus exhibiting an unusual replication cycle with genomic information going from RNA to DNA, prior to the integration of viral DNA into the host genome (Goff, 2007). Phylogenetic analyses placed HIV-1 in the lentivirus genus, alongside the previously described equine infectious anemia virus (EIAV), the caprine arthritis encephalitis virus (CAEV), the feline immunodeficiency virus (FIV) and the simian immunodeficiency virus (SIV). Lentiviruses are known to cause slow, chronic diseases in their respective hosts by targeting cells of the hematopoietic lineage, in particular lymphocytes and macrophages (Freed and Martin, 2007). Lentiviruses have a significantly more complex genomic organization than the so-called simple retroviruses, such as mouse mammary tumor virus (MMTV) or murine leukemia virus (MLV) (Goff, 2007). As a consequence, in addition to the usual structural and enzymatic proteins common to all retroviruses, HIV-1 encodes six specific auxiliary proteins crucial to modulate the cellular and systemic environment of the host (Fig. 3B) (Freed and Martin, 2007). Emergence of HIV in the human population was confirmed to occur through three independent interspecies transmissions of SIV from

16

(18)

chimpanzees (Gurtler et al., 1994; Peeters et al., 1997; Simon et al., 1998). Of the resulting genetically divergent HIV-1 M, N and O lineages, the M group accounts for the majority of cases among HIV-positive individuals (Kuritzkes and Walker, 2007). It is of interest to note that most SIV infections are asymptomatic in their natural host. Assuming a constant rate of evolution, introduction of HIV-I into the human population as been narrowed to the early 30’s, a very recent event in evolutionary terms (Korber et al., 2000). HIV-1 and humans therefore just started to co- evolve, a potential explanation of the current lethality of HIV-1 in its “new” host (Telenti, 2005).

5c. HIV-1 replication cycle

HIV-1 virion morphology

Mature HIV-1 virions are spherical particles, about 100-120 µm in diameter, delimited by a lipid bilayer of cellular origin harboring trimers of the viral envelope glycoprotein (Env) (Fig. 3A) (Freed and Martin, 2007). The Env viral protein results from non-covalent interaction between the gp120 and gp41 subunits, both cleavage products of the gp160 Env protein precursor (Freed and Martin, 2007). Beneath the lipid bilayer, a lattice of viral matrix protein (MA) surrounds an electron-dense conical core found at the center of mature particles. The outer layer of the viral core is composed of assembled viral capsid protein (CA) monomers while its innermost part contains two copies of genomic RNA tightly encapsidated in viral nucleocapsid proteins (NC) and associated with the viral reverse transcriptase (RT) and integrase (IN) (Fig. 3A). The exact location of the remaining viral proteins, i.e. the viral protease (PR), the auxiliary viral proteins Tat and Rev as well as the so-called accessory proteins Vif, Vpr, Vpu and Nef, is by and large unknown.

(19)

Env(gp120+gp41) MA CA NC p6 RT IN PR Vpr Vif Vpu Nef viral RNA genome

5’ LTR _gag 3’ LTR

pol

vif

env vpr

vpu nef

rev tat

MA CA NC p6

SP1 SP2

PR RT IN

U3 R U5 U3 R U5

gp120 gp41

A.

B.

Figure 3. Structure of the HIV-1 virion and the viral genome. (A) Schematic representation of a mature particle. Of note, the exact position of the viral non-structural proteins are yet to precisely be defined. (B) HIV-1 genomic organization and the resulting three polyproteins. All reminding viral proteins are synthesized as single proteins. Env:

envelope, MA: matrix, CA: capsid, NC: nucleocapsid, RT: reverse transcriptase, IN: integrase, PR: protease.

Viral entry

The HIV-1 replication cycle starts with adsorption of mature viral particles to the surface of susceptible cells (Fig. 4). CD4 receptor binding by the gp120 subunit of Env is crucial to the infectivity of HIV-1 and single-handedly defines its cell-tropism (Freed and Martin, 2007). CD4 binding, not only attaches HIV-1 virions to target cells, but is also a prerequisite that induces Env conformational modifications required for fusion at the plasma membrane and subsequent entry into target cells (Choe et al., 1996; Deng et al., 1996; Feng et al., 1996). The resulting additional interactions with the chemokine receptors CXCR4 or CCR5 trigger the fusogenic capacity of gp41 and fusion per se (Doms and Trono, 2000; Sattentau and Moore, 1991). HIV-1 strains are currently classified as R5-, X4- or R5/X4-viruses according to co-receptor usage (CCR5, CXCR4 or both, respectively) (Berger et al., 1998; Doms and Trono, 2000). R5-viruses are the predominant form of HIV-1 detected in recently infected individuals and remains so during most of the infection course (Zhu et al., 1993). Apparition and complete dominance of X4-viruses occurs mostly during the late symptomatic phase of the infection and is a serious indicator of rapid depletion of CD4⁺ T cells and progression towards AIDS (Connor et al., 1993; Koot et al., 1993).

18

(20)

receptor &

co‐receptor binding

fusion & entry

uncoating

reverse transcritpion

nuclear import

integration transcription Tat

Rev

Env synthesis & transport through the ER & Golgi apparatus viral proteins synthesis

PIC

Gag multimerisation & viral genomic RNA recruitment

plasma membrane targeting budding

maturation

Figure 4. HIV-1 replication cycle. Details are to be found in the appropriate sections of this text. Relevant literature (Freed and Martin, 2007) will further precise the many processes at play.

Reverse transcription and integration

Post-entry uncoating releases HIV-1 viral cores into the cytoplasm of the newly infected cell (Fig. 4). Sometimes called viral reverse transcription complexes, these subviral particles consist mainly of the genomic RNA, the viral RT and IN, as well as other viral and cellular components (Greene and Peterlin, 2002). It is within these complexes that the family defining reverse transcription step takes place. RT initiates minus-strand DNA synthesis from the tRNA^lys bound to the primer binding site (PBS) found on the genomic RNA. Concomitantly to DNA synthesis towards the 5’ en of the genome, the RNase H activity of RT digests the RNA component of the newly formed DNA/RNA complex, releasing the minus-strand strong-stop DNA intermediate (Fig. 5). Thanks to a short region of homology found at both end of the viral RNA genome (namely the R region), the minus-strand strong-stop DNA translocates to the 3’ end of the same genomic RNA. After strand transfer, RT rekindles the elongation of the minus-strand DNA, leading to tRNA^lys displacement. The simultaneous incomplete degradation of the RNA template leaves RNA fragments behind. Among those, the polypurine tract (PPT) is of particular importance and serves as primer for plus-strand DNA synthesis until a portion of the tRNA^lys is

(21)

reverse transcribed. Once again, RNase H activity of RT comes into play and digests the tRNA^lys duplexed with the newly synthesized plus-strand DNA, exposing its PBS at the 3’ end. This induces plus-strand DNA to jump to the homologous region at the 3’ end of the minus-strand DNA. The end product of plus- and minus-strand synthesis is a double-stranded viral DNA, with long terminal repeats (LTR) present at both ends (Fig. 5). LTR are multifunctional cis-acting sequences involved in integration and regulation of viral RNA synthesis notably. It is of interest to note that LTR per se are only present in the viral DNA and are not to be found on the genomic RNA (Freed and Martin, 2007). The end product of such intra molecular gymnastics is the HIV-1 preintegration complex (PIC) (Fig. 4). Composed of double-stranded viral cDNA, the viral IN, RT, MA, Vpr and NC proteins along with cellular factors, the PIC is responsible for targeting HIV-1 genomes to the host cell nucleus (Freed and Martin, 2007; Greene and Peterlin, 2002;

Miller et al., 1997). In contrast to other retroviruses, lentiviruses are able to infect nondividing cells. The PIC of HIV-1 must therefore possess means to cross the nuclear membrane and so in an active manner, its size preventing it from passively diffusing through nuclear pores. Of three viral proteins described so far to be involved in this process, only MA sports a standard nuclear location signal (NLS) recognized by components of the nuclear-import pathway (Bukrinsky et al., 1993). Although they lack canonical NLS, IN and Vpr are potential PIC components involved in nuclear transport (Gallay et al., 1997; Heinzinger et al., 1994). In addition, an intermediate product of reverse transcription, know as DNA flap because of a triple-helical DNA domain, might also be responsible for translocation into the nucleus by binding to cellular proteins containing a NLS (Zennou et al., 2000). The exact contributions of each protagonist in this process is however still confusing and remains to be clarified (Freed and Martin, 2007).

Integration as such is catalyzed by IN. After removing two nucleotides from each viral LTR, IN promotes the following joining reaction that sees the free viral cDNA turn into an integrated provirus in the host chromosome. Subsequently, the host cellular repair machinery fills the gaps created during the process, hence terminating integration (Greene and Peterlin, 2002).

20

(22)

Figure 5. From RNA to DNA: the reverse transcription. A substantial description can be found in the text. From (Coffin et al., 1997a).

Viral gene expression

HIV-1 proviruses are found at different chromosomal locations throughout the genome of infected T cells and either lead to latent or transcriptionally active integration events. In this latter case, transcription of the HIV-1 provirus is performed by the cellular RNA polymerase II (RNApol II) and leads to high levels of viral messenger and genomic RNA. In this context, the 5’

LTR functions like a regular transcriptional unit and provides a platform to recruit the RNApol II holoenzyme thanks to common promoter element such as the TATA box or Sp1 sites (Greene and Peterlin, 2002). Sequences upstream serve as a transcriptional enhancer and recruit the nuclear factor κB (NF-κB) and the nuclear factor of activated T cells (NFAT), leading to the initiation of transcription. In this setting however, RNApol II fails to elongate the viral RNA. To overcome this hurdle, the viral transactivator protein (Tat), in association with cyclin T1, binds the transactivation response (TAR) element, an RNA stem loop present on aborted viral RNAs, and in turn recruits the cellular cyclin-dependent kinase 9 (Cdk9), an integral member of the positive transcription-elongation factor b (P-TEFb) complex (Wei et al., 1998). Once recruited, Cdk9 phosphorylates the C-terminal domain of RNApol II, strongly increasing the processivity of the

(23)

polymerase and therefore viral RNA levels (Freed and Martin, 2007). Before export to the cytoplasm, postranscriptional modifications on viral RNAs are performed by the cellular processing machinery. But unlike most eukaryotic mRNAs, some HIV-1 mRNA must be exported unspliced (the genomic RNA, which will also serve as a mRNA for the Gag and Gag- Pol polyproteins), partially spliced (Env, Vif and Vpr mRNAs) or multiply spliced (Nef, Tat and Rev mRNAs). Incomplete splicing stems from the incorporation into HIV-1 transcripts of poor splice-donor and acceptor sites combined with the action of the regulator of expression of viral proteins (Rev). Rev, an RNA-binding protein, recognizes the Rev-responsive element (RRE) present in env and activates the export of unspliced or partially spliced viral mRNAs through interactions with Crm1, a member of the importin-β family involved in nuclear export (Freed and Martin, 2007). Expressed early from a multiply spliced mRNA, increasing concentrations of Rev protein tip the balance towards the export (and latter expression) of incompletely spliced viral transcripts, encoding the structural, enzymatic and most accessory viral proteins (Cullen, 1998).

Viral particle assembly

HIV-1 genome-length unspliced viral transcripts give rise to the viral polyprotein precursors Gag and Gag-Pol which, by and large, direct assembly and budding of newly synthesized virions (Fig.

4). The different domains of Gag and Gag-Pol play significant roles in the various steps leading to HIV budding and egress from infected cells. Gag and Gag-Pol polyproteins multimerize through protein-protein contacts between their respective CA domains (Freed, 1998) and recruit two copies of the viral genome through the interaction of their NC domain with the ψ site, a specific viral RNA packaging structure present just downstream of the 5’ LTR (Freed, 1998). Targeting of Gag-RNA complexes to the inner leaflet of the plasma membrane depends on the myristoilated moiety found at the N-terminal part of the MA domain (Bryant and Ratner, 1990; Gottlinger et al., 1989). Association with the plasma membrane is observed in cholesterol- and glycolipid- enriched membrane domains, the lipid rafts, thought to serve as viral assembly platforms stabilizing Gag-membrane binding (Ono and Freed, 2005). In addition, budding through these plasma membrane domains is thought to promote HIV-1 release, virion stability and its subsequent fusogenic capacity (Campbell et al., 2001). In parallel, the Env polyprotein gp160 is synthesized into the endoplasmic reticulum (ER), cleaved in the Golgi apparatus and brought to the cell surface through the secretory pathway (Fig. 4). Incorporation of the mature subunits of Env, gp120 and gp41, occurs at the time of viral budding and is mediated through direct interactions between Env and the MA domain of Gag and Gag-Pol multimers. Concurrently, HIV-1 virions passively inherit a number of cellular surface proteins, such as the human

22

(24)

leukocyte antigen (HLA) -I and –II, the lymphocyte function-associated antigen-1 (LFA-1) and the intercellular adhesion molecule-1 (ICAM-1) (Freed and Martin, 2007). Accumulation of Gag and Gag-Pol multimers at the plasma membrane self-induces membrane curvature and promotes the formation of immature HIV-1 particles still tethered to the cell surface, their Gag and Gag-Pol precursors radially organized with their N-termini towards the membrane and their C-termini towards the center (Fig. 4 and 6) (Freed and Martin, 2007). The final step in HIV-1 budding involves pinching off of newly synthesized virions from the plasma membrane and depends on a last domain of the Gag precursor, namely the p6 domain (Fig. 6c). Through a PTAP motif, p6 interacts with the tumor suppressor gene 1 (Tsg101), a member of the endosomal sorting complex required for transport-I (ESCRT-I). Through this binding, HIV-1 diverts to its benefit an entire cellular machinery usually involved in budding of vesicles into the lumen of the late endosome/multivesicular body (MVB) (Fig. 14B) (Garrus et al., 2001; Katzmann et al., 2001;

VerPlank et al., 2001). Upon or directly after viral release from the plasma membrane, self- cleavage of the PR from the Gag-Pol polyprotein precursor initiates maturation of HIV-1 particles (Pettit et al., 2004). During this process, the Gag and Gag-Pol precursors are processed by the PR into their respective constituents, profoundly modifying the organization of the virions into electron-dense particles with a condensed conical core (Fig. 6d) (Bukrinskaya, 2004; Freed and Martin, 2007). While a wealth of morphological data has made budding from the plasma membrane the “classical” model for HIV-1 egress, some HIV-1-permissive cell types (i.e macrophages) exhibit assembly and budding of newly synthesized virions in an intracellular compartment (Fig. 14A) (Orenstein et al., 1988; Pelchen-Matthews et al., 2003; Sharova et al., 2005). Recent studies in macrophages would tend to reconcile the two views as plasma membrane invaginations, with an unexpected level of complexity, seem to be the source of the HIV-1- containing compartment in these cells (Deneka et al., 2007; Jouve et al., 2007; Welsch et al., 2007). Because this particular aspect of HIV-1 biology has been of vivid interest during my thesis, further details are to be found in the corresponding sections of this manuscript.

c.

a. b. d.

Figure 6. HIV-1 budding and maturation. The dense accumulation of Gag and Gag-Pol poly proteins to the inner leaflet of the plasma membrane induce membrane curvature (a, b) until the budding viral particle pinches off in the extracellular milieu. Upon or right after budding, maturation of the viral particle occurs leading to a morphological rearrangement and condensation of the capsid. From (Coffin et al., 1997b). Original images from(Swanstrom et al., 1990).

(25)

Accessory proteins

In addition to structural, enzymatic and regulatory proteins, HIV-1 genome encodes 4 different proteins misleadingly dubbed “accessory” proteins (Fig. 3B). Although often dispensable for viral replication in cell culture, tissue culture and in vivo experiments have shone a new light on these proteins, revealing their importance for efficient viral infection (Freed and Martin, 2007).

- Vpr: Among these four accessory proteins, Vpr is significantly incorporated into virions through its interaction with the p6 domain of Gag (Kondo and Gottlinger, 1996; Lu et al., 1995;

Yuan et al., 1990). Although it contributes poorly to the replicative fitness of HIV-1 in proliferating T-cell cultures, Vpr plays a substantial role in non-dividing monocyte-derived macrophages (MDM) (Freed and Martin, 2007). Data gathered from Vpr-deficient HIV-1 strains isolated from long term nonprogressors as well as from modestly pathogenic Vpr-deleted SIV in rhesus macaques infection experiments indicate a significant role for Vpr in vivo (Lang et al., 1993; Somasundaran et al., 2002). As mentioned previously in this manuscript, the presence of Vpr in the PIC of HIV-1 and in the nucleus of infected cells strongly suggests a role for this protein in the nuclear import of viral components (Heinzinger et al., 1994; Lu et al., 1993).

Whether the mechanisms involved in this process are dependent or independent from the importin-β nuclear import machinery remain however to be clarified (Freed and Martin, 2007).

Vpr has also been shown to mildly induce gene expression from the HIV-1 LTR and to induce cell-cycle arrest in the G2 phase (Cohen et al., 1990; Poon et al., 1998). As HIV-1 transcription occurs mainly during the G2 phase, the cell-cycle arrest function of Vpr might therefore provide a favorable nuclear environment to HIV-1 (Goh et al., 1998). An additional role in apoptosis has been proposed for Vpr, a role for which the description of the exact mechanisms are ongoing (Andersen et al., 2006; Muthumani et al., 2005; Schrofelbauer et al., 2007).

- Vpu: Unique to HIV-1 and the related SIVcpz, the viral protein U (Vpu) is an integral membrane protein involved in CD4 degradation and enhancement of viral particle release. Proteasomal degradation of CD4 occurs after Vpu simultaneous binding, in the ER, to CD4 and to the cellular factor targeting proteins to the proteasome, β-TrCP (Freed and Martin, 2007). Degradation of CD4 liberates the Env precursor gp160 and allows Env to proceed to the cell surface, increasing HIV-1 particles infectivity (Willey et al., 1992a; Willey et al., 1992b). In parallel, Vpu enhances HIV-1 particle release by counteracting the effects of a cellular factor promoting the internalization of newly budded virions and hence restricting particle release (Neil et al., 2006).

24

(26)

This cellular inhibitor of virus release has been recently identified as CD317 (or tetherin) (Neil et al., 2008; Van Damme et al., 2008). Due to the homology between the trans-membrane domain of Vpu and the K⁺ channel protein TASK-1, it has also been envisioned that Vpu might enhance viral release by inhibiting cellular ion channel activity (Freed and Martin, 2007).

- Nef: Nef was first described as diminishing viral replication in infected cells and hence was erroneously labeled viral negative factor. Anchored to the inner leaflet of the plasma membrane thanks to its myristoilated N-terminus (Welker et al., 1998), HIV-1 Nef has been described to downregulate several cell-surface molecules, such as CD4, MHC-I and MHC-II (Piguet et al., 1998; Schwartz et al., 1996; Stumptner-Cuvelette et al., 2001). In the case of CD4 and MHC-I, Nef binds directly to the cytoplasmic domain of the proteins and targets them, in conjunction with other cellular partners, to lysosomal degradation. In addition, Nef downmodulates the expression of MHC-I as well as MHC-II preventing their accumulation at the cell surface. The reduced presence of CD4 on the cell surface avoids superinfection, favors HIV-1 release from the cell surface as well as proper Env incorporation (Lama et al., 1999; Ross et al., 1999). Low cell- surface levels of MHC-I are certainly a strong advantage in avoiding the CTL-mediated killing of HIV-1-infected cells (Collins et al., 1998). The concomitant poor MHC-II restricted peptide presentation further impairs a proper anti-HIV-1 immune response by preventing the full activation of HIV-1-specific CD4⁺ T cells (Freed and Martin, 2007). Furthermore, Nef interferes with cellular signal transduction pathways and modulates T-cell activation, a cellular state more propitious to HIV-1 replication (Fenard et al., 2005; Krautkramer et al., 2004). Finally, HIV-1 infectivity is also mildly increased by Nef, a process yet unclear but certainly linked to the association of Nef with the viral core during the early postentry steps (Campbell et al., 2004).

- Vif: The viral infectivity factor (Vif), crucial for HIV replication, is produced to high levels in producer cells. Although elusive until recently, the crucial role of Vif has finally found an answer with the discovery of the antiretroviral cellular restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) (Harris et al., 2003). APOBEC3G is a cellular DNA cytidine deaminase that induces G-to-A hypermutations of the viral genome of Vif- deficient HIV-1 virions (Lecossier et al., 2003; Mangeat et al., 2003; Zhang et al., 2003).

Additional studies have led to the understanding that Vif has evolved to counteract the deaminating activity of APOBEC3G by targeting the restriction factor for degradation through the ubiquitin-proteasome pathway (Mehle et al., 2004; Yu et al., 2003).

(27)

5d. Dendritic cells

The routes of HIV-1 transmission are varied and reflect the presence of viral particles in several body fluids such as blood, cerebrospinal fluid, vaginal secretion and semen. Because of high numbers of infectious HIV-1 particles in blood, needle sharing among drug users and blood- related medical acts are still serious dissemination modes in western and in developing countries respectively. Vertical HIV-1 transmission from mother to child can occur through the placental barrier but is however more likely to take place during delivery as a result from exposure to infected vaginal secretions and genital tract. Fortunately, mother-to-child transmission has been decreasing, at least in western countries, due to antiviral drug therapy administration during pregnancy. In a global perspective, the number one route of HIV-1 transmission worldwide results from unprotected sexual intercourse (Flint et al., 2004). Through this route, HIV-1 enters the host through mucosal surfaces before spreading throughout the body. Among the various cells present at the portal of entry, dendritic cells (DC) associated with exposed mucosal surfaces represent the first line of defense against incoming virions. Sadly, HIV-1 seems to have found a way around its opponent, turning DC into potentially one of its strongest allies.

DC as ‛professional’ antigen presenting cells

DC are a diverse cellular population central to immunity. Along with other antigen presenting cells (APC), such as B cells and macrophages, they strongly contribute to a proper, controlled and efficient T cell response to invading pathogens (Banchereau and Steinman, 1998). As their collective name implies, APC do so through the presentation on their cell surface of foreign antigens recognized by T lymphocytes. Schematically, this process results from antigen uptake, its partial degradation and loading onto major histocompatibility complex (MHC) class I and II molecules and its presentation to T cells. All APC are not however equivalent and their respective function only marginally overlap. B cells, while limited in their endocytic ability, present antigens to T cells but, unlike DC, do not facilitate T cell responses per se. B cell-antigen presentation is nevertheless essential as it participates to the onset of the humoral arm of the immune response (Trombetta and Mellman, 2005). Conversely, although macrophages exhibit an acute capacity for endocytosis, relative low levels of MHC-I and -II on their cell surface makes them poor antigen presenters compared to DC. In addition, the impressive endocytic machinery of macrophages is clearly geared towards pathogen clearance, making proper antigen processing and presentation a subsidiary task (Trombetta and Mellman, 2005). Therefore, DC set themselves apart from other APC by focusing on antigen presentation. Present in most peripheral tissues, DC scan their direct

26

(28)

environment and serve as sentinels to the immune system. In the absence of pathogenic threats, a fraction of DC constitutively circulates through peripheral tissues, the lymph and secondary lymphoid organs, reaching T cell-rich areas. Steady-state levels of DC in peripheral tissues is maintained through migration in the tissues of DC-precursors, present in the blood (Banchereau and Steinman, 1998).

Considerably more competent at T cell stimulation than any other APC, DC are able to activate immunologically naïve T cells, drive their proliferation and differentiation, and therefore instigate the adaptive immune response (Banchereau and Steinman, 1998; Lanzavecchia and Sallusto, 2001b). DC do so by establishing specific cell-cell contacts with T cells called immunological synapses (Bromley et al., 2001; Grakoui et al., 1999). Within these structures, T cell receptors (TCR) recognize antigenic peptides-MHC (p-MHC) complexes. Multiple TCR triggering by p- MHC complexes induce signaling in the T cell and, according to the amount of signal received, determine the fate of the T cell (Lanzavecchia and Sallusto, 2001b). The intensity of the signaling cascade induced in T cells highly depends on the amounts of p-MHC complexes present in the immunological synapse from the start. Subsequent engagement of DC co-stimulatory molecules (i.e. CD86) by the T cell co-stimulatory receptor (CD28) further amplifies the amount of signal.

Finally, the duration of the DC-T cell interaction defines the amount of time where signal transduction occurs (Lanzavecchia and Sallusto, 2001a). Although relatively stable, immunological synapse formation and immunological synapses themselves must be envisioned as rather dynamic processes, where T cells sequentially engage in multiple immunological synapses with different DC (Mempel et al., 2004; Stoll et al., 2002). In parallel to robust T cell activation, DC also qualitatively tailor the immune response by polarizing T cell development or by inducing peripheral T cell tolerance to tissue-specific antigens (Lanzavecchia and Sallusto, 2001b).

The endocytic ability of DC

DC mediate antigen uptake through the endocytosis of whole pathogens, infected and dead cells or their derived products. Antigens enter the endocytic pathway through three mechanisms:

phagocytosis, macropinocytosis and receptor-mediated endocytosis (Fig. 7) (Banchereau and Steinman, 1998). Phagocytosis, a receptor-mediated, actin-dependent process, allows DC to efficiently engulf large intact microbes (i.e. bacteria) or apoptotic cells. Phagocytosis of the later in the context of microbial infections emerges as a crucial mean for DC to screen dying cells for potentially hidden pathogens, especially when DC themselves do not get infected (Carbone and Heath, 2003). In addition, DC are capable of sampling large amounts of extra-cellular fluids for antigens through macropinocytosis. Although unspecific, this actin-based process allows DC to

(29)

rapidly sample their environment for soluble antigens derived from infection (Guermonprez et al., 2002). Whether macropinocytosis plays a central role in vivo, where DC encounter antigens mostly in tissues with little extracellular fluid present, remains to be fully assessed (Trombetta and Mellman, 2005). Through the expression of a wide variety of cells surface receptors, DC are able to internalize antigen in a more specific manner. Receptor-mediated endocytosis renders antigen uptake very efficient by a concentrating effect, making only minute quantities of antigen sufficient to induce a vigorous immune response (Banchereau and Steinman, 1998). Moreover, this pathway provides also the immune system with a potential tool to qualitatively adjust its responses to diverse threatening situations through the differential expression of various receptors on different DC subsets (Trombetta and Mellman, 2005). Among receptors involved in antigen- uptake, such as Fc receptors and scavenger receptors, the C-type lectin family of receptors (CLR) is of particular interest. C-type lectins bind their ligands through a common carbohydrate recognition domain (CRD), in a Ca⁺⁺-dependent manner. DC express a wide variety of these lectins like the macrophage-mannose receptor (MMR), DEC-205, the DC-specific ICAM-3- grabbing nonintegrin (DC-SIGN) and Langerin (Guermonprez et al., 2002). Endocytosis of CLR is entirely dependent on their intracellular internalization motifs and may provide differential ways to respond to antigens. Many pathogens target these receptors and seem to hinder their function (van Kooyk et al., 2004). DC-SIGN, present on DC, was shown to bind HIV-1 and favor its infection of co-cultured T cells, a process termed trans-infection (Geijtenbeek et al., 2000b).

Because DC-SIGN/HIV-1 interactions have gathered my attention during my thesis, further details are to be found in the corresponding section of this manuscript.

clathrin

Figure 7. Endocytosis in DC. Phagocytosis, macropinocytosis and receptor-mediated endocytosis are the main routes of antigen uptake in DC. Note that the later can also occur in a clathrin-independent manner. Adapted from (Trombetta and Mellman, 2005).

28

(30)

Antigen presentation in DC

In DC, like in all APC, antigens are processed into peptides and presented as such in association with MHC molecules. A classical and simplistic view of antigen presentation would have antigenic peptides of endogenous and exogenous origin loaded onto MHC-I and MHC-II molecules, respectively. The resulting p-MHC complexes then respectively serve as recognition signals for priming CD8⁺ and CD4⁺ T cells responses. Although true to some extent, the situation is indeed slightly more complex with MHC-I and –II molecules able to associate with both endo- and exogenous antigens (Guermonprez et al., 2002). Regardless, most peptides loaded on MHC-I are indeed of endogenous origin. In this case, antigen degradation involves the proteasome and therefore implicates ubiquitination of proteins present in the cytosol. Then, thanks to the specific peptide transporter TAP, peptides cross the ER membrane, are loaded on MHC-I molecules before p-MHC-I complexes are brought to the cell surface through the Golgi apparatus. In the context of viral infection, this antigen-presenting pathway allows the generation of a virus- specific CTL-response only if DC themselves are infected. This is however not always the case and DC must resort to cross-presentation in order to mount a proper cell-mediated immune response. This process occurs mostly in a TAP-dependent manner, implying that exogenous antigens reach the cytosol from the endocytic pathway in a yet to be defined fashion. Taking advantage of the marginal presence of MHC-I molecules in endocytic compartments, minor cross-presentation takes place also in a TAP-independent manner (Guermonprez et al., 2002;

Trombetta and Mellman, 2005). In the case of MHC-II molecules, antigen loading occurs at a different intracellular location. After synthesis, MHC-II components traffic through the ER and the Golgi apparatus before being transported to the endocytic pathway where antigenic peptides are loaded. p-MHC-II complexes are then transported to the cell surface where they are recognized by cognate CD4⁺ T cells. Internalized exogenous proteins as well as endogenous plasma membrane and endosomal proteins, cleaved by several proteases of the cathepsin family, are the source of the antigenic peptides presented on MHC-II molecules (Guermonprez et al., 2002; Trombetta and Mellman, 2005). However, antigens present in the cytosol or in the nucleus can also be found associated with MHC-II molecules, despite of the seemingly topological segregation. Transfer of cytosolic antigens to the endocytic pathway seems to occur at the levels of lysosomes by a process called autophagy. This pathway allows cells to digest parts of their cytosol in order to recycle nutrients or dispose of unnecessary cytoplasmic components and unwanted cytoplasmic pathogens. In addition, autophagy links cytoplasmic antigens with MHC-II presentation (Levine and Deretic, 2007). It is important to note that DC harbor a third kind of MHC molecules involved mainly in glycolipids presentation to T cells. These molecules, the CD1

Characterization of a Novel Tetraspanin-Rich, HIV-1-Containing Compartment in Dendritic Cells

Thesis

Reference