HIV-1 innate immune detection and evasion

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Thesis

Reference

HIV-1 innate immune detection and evasion

REINHARD, Christian

Abstract

Dendritic cells are important sentinels of the innate immune system that present antigen to the adaptive immune system, initiating the host immune response. Dendritic cells can be infected by HIV-1, but, in the absence of productive infection dendritic cells facilitate virus transfer to highly permissive CD4+ T cells. HIV-1 is sensitive to the antiviral state induced by type I interferon but the virus is able to evade detection by pattern recognition receptors (PRR) and thereby avoids the induction of a robust immune response. To understand the interactions between HIV-1 and the innate immune system we focused on two host factors (TRIM5 and SAMHD1). TRIM5 is a restriction factor that binds the retroviral capsid lattice and blocks viral replication. We showed that TRIM5 is also a signal transducer in the type I interferon pathway.

Using a newly developed method to knockdown genes of interest in monocyte derived dendritic cells (MDDCs), we demonstrated that TRIM5 knockdown cells produce less type I interferon in response to LPS, which in turn leads to a weaker antiviral state in the TRIM5 knockdown cells, as compared to the [...]

REINHARD, Christian. HIV-1 innate immune detection and evasion. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4610

URN : urn:nbn:ch:unige-318897

DOI : 10.13097/archive-ouverte/unige:31889

Available at:

http://archive-ouverte.unige.ch/unige:31889

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

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UNIVERSITÉ DE GENÈVE

Département de biologie moléculaire FACULTÉ DES SCIENCES Professeur Robbie Loewith

Département de microbiologie FACULTÉ DE MÉDECINE

et médecine moléculaire

Professeur Jeremy Luban

HIV-1 Innate Immune Detection And Evasion

THESE

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

Par

Christian REINHARD Sumiswald (BE) De

Thèse n° 4610

Genève 2013

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UNIVERSITE DE GENEVE

F A C U L T E D E s S C I E N C T S

Docloro] ds sciences Mention biologie

Thdse ae lllonsieur Christian REINHARD

i n t i t u l 6 e :

" HIV-I lnnofe lmmune Deleclion And Evosion "

Lo Focult6 des sciences, sur le pr6ovis de Messieurs J. LUBAN, professeur et directeur de thdse (Focult6 de m6decine, D6portement de microbiologie et medecine mol6culoire), R. LOEWITH, professeur ossoci6 et codirecteur de thdse (D6portement de biologie mol6culoire), D. GARCIN, docteur (Focult6 de m6decine, D6portement de microbiologie et m6decine mol6culoire) et O. SCHWARTZ, docteur (lnstitut Posteur, Unit6 Virus et lmmunit6, Poris, Fronce), outorise I'impression de lo pr6sente thdse, sons exprimer d'opinion sur les propositions qui y sont 6nonc6es.

G e n d v e , l e l 6 o c t o b r e 2 0 1 3

Thdse - 461 0 -

L o t h e s e d o i t p o r t e r dons les "lnformotions

l e s c o n d i t i o n s 6 n u m 6 1 6 e s I ' U n i v e r s i t 6 d e G e n d v e " .

Doyen,

-Morc TRISCONE

N . B . lo d6clorotion pr6c6dente et remplir r e l o t i v e s o u x t h d s e s d e d o c t o r o t d

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“Non est ad astra mollis e terris via“

Seneca

It was a long journey which led to this point, where I am allowed to call myself Doctor.

When I started as a PhD-student I did not know what it meant but I knew that I wanted to become a scientist. For giving me the opportunity to do my thesis in his lab, I am very grateful to Prof. Jeremy Luban. I will never forget all the fruitful discussions, which always led to new ideas and projects. I learned a great deal from him, professionally and personally.

Looking back over the last four years I realize that a doctorate is much more than collecting enough data to write a thesis. It is about becoming a member of the academic community, being able to think independently, to come up with new hypothesis and novel ways to test them and of course reject most of them. Failed experiments and projects are a big part of scientific research but because of all the failed ones the successful experiments taste so much sweeter. “In face of repeated failure you can prove that you are a true scientist by going on”, Massimo Pizzato told me that. He is an amazing mentor, role model and friend.

Because of the people I met during my time in Geneva my PhD became such a memorable experience. For that I will be always thankful to them. First of all: Yoselin Grimaldi, love of my life. Alberto De Iaco is a great friend and made always sure that we had fun. Jessica Guerra and Dario Bottinelli, we made a great team in the lab and outside.

Stéphane Hausmann, Josefina Lascano and Madeleine Zufferey were wonderful colleagues.

I am very grateful that Dr. Dominique Garcin and Prof. Laurent Roux adopted me scientifically in my lonely last year and that Strubin lab adopted me for many lunches and TGIFs.

I am also grateful to Prof. Robbie Loewith, Prof. Olivier Schwartz and Dr. Dominique Garcin to be my PhD jury. It was a pleasure to discuss my thesis with them.

Many thanks also go to my parents Esther and Hansueli, who were always there to support and help me when I needed them. To my brother Benjamin, his wife Martina and especially to my little niece Elin: Her smile makes me feel happier than a thousand successful experiments. To my sister Myriam and her husband Bruno, a trip to beautiful Grindelwald was always a welcome change of scenery.

Christian Reinhard

Geneva, 31^th October 2013

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Abstract

Dendritic cells are important sentinels of the innate immune system that present antigen to the adaptive immune system, initiating the host immune response. Dendritic cells can be infected by HIV-1, but, in the absence of productive infection dendritic cells facilitate virus transfer to highly permissive CD4⁺ T cells. HIV-1 is sensitive to the antiviral state induced by type I interferon but the virus is able to evade detection by pattern recognition receptors (PRR) and thereby avoids the induction of a robust immune response. To understand the interactions between HIV-1 and the innate immune system we focused on two host factors (TRIM5 and SAMHD1).

TRIM5 is a restriction factor that binds the retroviral capsid lattice and blocks viral replication. We showed that TRIM5 is also a signal transducer in the type I interferon pathway. Using a newly developed method to knockdown genes of interest in monocyte derived dendritic cells (MDDCs), we demonstrated that TRIM5 knockdown cells produce less type I interferon in response to LPS, which in turn leads to a weaker antiviral state in the TRIM5 knockdown cells, as compared to the control cells. The induction of inflammatory mRNA level and secreted proteins was also reduced in TRIM5 knockdown.

Challenge with capsid variants that are restricted by TRIM5 stimulated the production of inflammatory cytokines, demonstrating that TRIM5 is a pattern recognition receptor specific for the retroviral capsid.

SIVMAC/HIV-2 accessory protein Vpx acts as an adaptor with DCAF1 to target the host protein SAMHD1 for ubiquitination and proteasomal degradation. Disruption of SAMHD1 in myeloid and resting T cells leads to an increase in the intracellular nucleotide pool, permitting reverse transcription to take place that it would otherwise be blocked in the presence of SAMHD1. We showed that Vpx rescues HIV-1 from it mature MDDCs but it does not rescue SIVMAC or HIV-2. This rescue of HIV-1 is independent of DCAF1 and the increase of the intracellular nucleotide pool. When compared to the effect of treating cells with exogenous nucleosides to artificially increase the intracellular nucleotide pool, Vpx increases nuclear import and integration of the HIV-1 genome in mature dendritic cells and monocytes.

Taken together the data obtained in these two projects clarifies the interaction between HIV-1 and the innate immune system. The first project elucidates how retroviruses are detected by the innate immune system. The second project sheds light on the mechanisms that HIV-1 and other retroviruses use to evade detection by the innate immune system and the associated activation of effectors that block viral replication.

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Résumé

Les cellules dendritiques jouent un rôle important comme sentinelles du système immunitaire inné et présentent l'antigène au système immunitaire adaptatif menant à l’initiation de la réponse immunitaire de l’hôte. Dans le contexte du VIH-1, les cellules dendritiques sont infectées et sont utilisées par le virus afin d'être transféré aux cellules T CD4⁺. VIH-1 est sensible à l'état antiviral induit par l’interféron de type I, mais ce virus est capable d’éviter la détection par des récepteurs qui reconnaissent des « motifs moléculaires associés aux pathogènes » et par conséquent évite d’induire une forte réponse immunitaire.

TRIM5α lie la capside rétrovirale et bloque la réplication virale. En plus de son rôle de facteur de restriction, nous montrons que TRIM5α est également un convertisseur de signal dans la voie de l'interféron de type I. En utilisant une nouvelle méthode pour supprimer l’expression de TRIM5α dans les cellules dendritiques dérivées de monocytes (MDDCs), nous avons démontré que les cellules n’exprimant pas TRIM5α produisent moins d’interféron de type I lorsqu’elles sont stimulées par du LPS. En conséquence, ces cellules induisent un état antiviral plus faible que des cellules témoins. Le même effet a été observé sur l'induction de cytokines pro-inflammatoires au niveau de l'ARNm et des protéines sécrétées. TRIM5α possède le rôle d'une nouvelle fonction en tant que récepteur de reconnaissance de formes pour la capside rétrovirale, car des variantes de la capside qui sont restreintes par TRIM5α ont stimulé la production de cytokines inflammatoires.

La protéine accessoire SIVMAC/HIV-2 Vpx vise le facteur de l'hôte SAMHD1 pour le dégrader par le protéasome. L'absence de SAMHD1, dans les cellules T et myéloïdes au repos, entraîne une augmentation du pool intracellulaire de nucléotides, permettant à la transcription inverse de prendre place quand elle serait autrement bloquée en présence de SAMHD1. Nous montrons que Vpx protège le VIH-1 de l'état antiviral dans les MDDCs mais cette protéine ne peut pas protéger SIVMAC ou le VIH-2. Cette protection est indépendante de l'augmentation du pool intracellulaire de nucléotides car Vpx est capable d'augmenter l'import nucléaire et l'intégration du génome du VIH-1, tandis que l’augmentation artificielle de la concentration nucléotidique intracellulaire augmente seulement le taux de transcription inverse.

Prises ensembles, les données présentées ici permettent de mieux comprendre comment les rétrovirus sont détectés par le système immunitaire intrinsèque et inné, et met en lumière le mécanisme utilisé par le VIH-1 et d'autres rétrovirus pour échapper à l'action du système immunitaire inné.

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List of Abbreviations

A3G APOBEC3G

ADAR1 adenosine deaminase acting on RNA 1 AGS Aicardi-Gutières Syndrome

AIDS acquired immunodeficiency syndrome BST-2 B cell stromal factor 2

CA capsid

CADRIF CARD adaptor inducing IFNβ cDCs conventional dendritic cells cDNA complementary DNA cGAS cyclic GMP-AMP synthase CMV cytomegalo virus

cPPT central polypurine tract CRL Cullin-RING finger E3 ligase CTD C-terminal domain

CTL cytotoxic T lymphocyte (CD8⁺ T cell) CypA cyclophilin A

DAMP danger-associated molecular pattern DCAF1 DDB1-and Cullin4-associated factor 1 DCs dendritic cells

DC-SIGN DC-specific ICAM3-grapping

DDB1 damage-specific DNA binding protein DIS dimer initiation signal

dNTP deoxyribonucleotide triphosphates dsDNA double stranded DNA

dsRNA double stranded RNA ER endoplasmic reticulum

ESCRT endosomal sorting complex required for transport FIV feline immunodeficiency virus

Fv1 Friend virus susceptibility 1 GALT gut associated lymphoid tissue

GM-SCF granulocyte/macrophage colony stimulating factor HAART highly active antiretroviral therapy

HIV human immunodeficiency virus HSC hemapoietic stem cells

IFN interferon IL interleukin

IN integrase

IRF IFN regulatory factor ISGF IFN stimulated gene factor ISGs interferon stimulated genes ISRE IFN-stimulated response element JAK1 Janus kinase 1

kb kilo bases

kDa kilodalton (molecular weight)

KD knockdown

LC Langerhans cells

LRT late reverse transcription product LTR long terminal repeat

LV lentiviral vector

MA matrix

MAPK mitogen-activated protein kinase MAVS mitochondrial antiviral protein

MDA5 melanoma differentiation-associated gene 5

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10 MDDCs monocyte derived dendritic cells MDMs monocyted derived macrophage MHC major histocompatibility complex MLV murine leukemia virus

NC nucleocapsid

Nef negative factor NES nuclear export signal

NFAT nuclear factor of activated T cells NF-κB nuclear factor κB

NLS nuclear localization signal NTD N-terminal domain

ORF open reading frame

PAMP pathogen-associated molecular pattern PBS primer binding site

PIC pre-integration complex PKR protein kinase R

PPT polypurine tract

PR protease

Pr160gag-pol 160 kDa Gag Pol precursor Pr55gag 55 kDa Gag precursor PRR pattern recognition receptor R short repeated sequence RIG-I retinoic acid-inducible gene I RRE Rev responsive element RT reversetranscriptase siRNA small interfering RNA

SIV simian immunodeficiency virus ssDNA single stranded DNA

ssRNA Single stranded RNA

STAT1 signal transducer and activation of transcription 1 STING stimulator of IFN genes

TAK1 TGFβ activated kinase 1

TAR transactivation response element TBK-1 TANK-binding kinase 1

TLR Toll-like receptor TNFα tumor necrosis factor α

TRAF TNF receptor associated factor TREX1 3’ prime repair exonuclease 1

TRIF Toll-IL1 resistance domain-containing adaptor inducing IFNβ TRIM tripartite motif containing protein

U3 unique 3’ sequence in the HIV-1 LTR U5 unique 5’ sequence in the HIV-1 LTR UPS ubiquitin-proteasome system

Vif viral infectivity factor VLPs virus like particles Vpu viral protein r Vpu viral protein u Vpx viral protein x

VSV vesicular stomatitis virus Ψ packaging signal

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Chapter 1: General Introduction

1.1 Three decades of HIV/AIDS

The first cases of acquired immunodeficiency syndrome (AIDS) were described in June 1981 in Morbidity and Mortality Weekly Report. Five young homosexual men with opportunistic Pneumocystis carinii pneumonia (PCP), extensive mucosal candidiasis and severe viral infections including cytomegalovirus infection (CMV) were described in Los Angeles [1]. Shortly afterwards 26 men in New York and California presented with Kaposi’s sarcoma and PCP [2]. It became clear that these men had a common immunological deficit in cell-mediated immunity caused by a depletion of CD4⁺ T cells causing an immune suppression and the occurrence of associated pathologies [3, 4]. In 1983 in the laboratory of Luc Montagnier at the Institute Pasteur in Paris, Françoise Barré-Sinoussi was able to isolate a T cell tropic retrovirus from a patient suffering from this immune suppression, which became known as acquired immunodeficiency syndrome (AIDS) [5]. In 1984 the French group presented together with the group of Robert C. Gallo at the US National Institute of Health evidence of the retrovirus causing AIDS later termed human immunodeficiency virus (HIV) [5-9]. The virus was also isolated from patients with AIDS independently by Jay Levy in San Francisco [10]. The early work led quickly to an HIV-1 test to detect the virus in the blood of infected people and blood donation screenings; decreasing the risk of infection via blood transfusion rapidly ([11] and reviewed here [12]). Françoise Barré-Sinoussi and Luc Montagnier were awarded the Nobel Prize for Physiology or Medicine in 2008.

Over thirty years after the outbreak of AIDS and the discovery of the causing agent, the disease still poses a major health and economical threat to the human population. According to the UNAIDS report issued by the World Health Organization (WHO) in 2012, 30.4 million people were estimated to live with HIV-1 in 2011. In 2005 2.3 million people still died from AIDS or AIDS related causes. Due to better treatment and prevention measures these numbers have been declining globally in recent years and in 2011 the number of people dying from AIDS has decreased 24%

to 1.7 million people. The number of newly infected patients has also decreased 20%

since 2001 and reached 2.5 million people per year worldwide in 2011 (UNAIDS report 2012). Despite the progress achieved in treating and preventing HIV infection,

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the search of the best preventive weapon against a virus – a vaccine – has still not yielded success.

1.2 The origin of the HIV pandemic

The HIV-1 pandemic spread over the entire globe but sub-Saharan Africa, where the pandemic started is still greatly affected. HIV originated from retroviruses of species-specific simian immunodeficiency virus (SIV) circulating in African monkey species. Several separate cross-species transmission from SIV infecting chimpanzees (Pan troglodytes troglodytes) SIVcpz [13] and from gorilla (Gorilla gorilla gorilla) SIVGOR [14] gave rise to four main HIV-1 groups: M “main”, O “outlier”, N

“non-M, non-O” and P “pending the identification of further human cases”. A virus from the group P has been discovered only recently in a woman from Cameroon. The virus was more closely related to SIVGOR than SIVCPZ [14]. The vast majority of patients are infected with M-group viruses, while N- and O-group virus infections are clustered around Cameroon and account for only a small proportion of infected patients. Group M is again subdivided into clades (A to K) with the most common clades being clade A found in West Africa, clade B dominant in Europe, the Americas, Australia, Japan and Thailand and clade C in Southern Africa and India.

In separate incidences SIVSM infecting sooty mangabey (Cercocebus atys atys) a non-primate monkey inhabiting west Africa, gave rise to HIV-2 when it jumped the species barrier [15]. These viruses are continued to be transmitted from their animal hosts to humans causing new zoonotic outbreaks [16]. Interestingly, while SIVSM and SIVCPZ are generally not causing AIDS in their natural host [17, 18], SIVSM causes severe AIDS in rhesus macaque monkey (Macaca mulatta) indigenous to Asia with no natural SIV infection [19]. The opinion that SIVCPZ infection causes no major pathologies was revised when a recent study of free-ranging chimpanzees discovered that infected females were less likely to give birth and had higher child mortality and clinical symptoms of end stage AIDS were observed in an infected individual [20].

1.3 Virion structure and genomic organization of HIV

HIV-1 classifies to the genus of lentivirus, which belongs to the family of retroviruses. It is an enveloped virus and carries two copies of single-stranded (ss) positive-strand RNA genome (+ssRNA). The name retrovirus stems from its ability to

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reverse transcribe the RNA genome via the viral reverse transcriptase (RT) – an RNA dependent DNA polymerase [21, 22] - into a double stranded complementary DNA (cDNA), which is permanently integrated by the viral integrase (IN) into the genome of the host cell [23].

The HIV-1 virus particle (virion) has a spherical shape and measures roughly 145 nm in diameter (Figure 1.1). The virion constitutes of a capsid containing the RNA genome wrapped by a lipid bilayer envelope derived from the host cell. Trimers of the viral envelope protein (Env) are imbedded into the envelope. Env consists of two non-covalently attached glycoproteins, the transmembrane protein gp41 is positioned in the viral envelope and anchors gp120 to the envelope. Putative neutralizing epitopes are shielded by neighboring gp120 molecules and heavy glycosylation [24].

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It has a high sequence diversity, which allows the virus to escape from neutralizing antibodies [25, 26]. Gp120 receptor binding induces a conformational change in gp41 exposing the fusion peptide, which gets inserted into the host cell plasma membrane [27] and triggers the fusion of the two membranes. Gp120 binds the main HIV-1 cellular receptor, the CD4 molecule mostly expressed on CD4⁺ T cells [28]. To trigger fusion with the target cell membrane, binding one of two co-receptors either CXCR4 [29] or CCR5 [30] is necessary as well. The two different co-receptors determine the tropism of the virus. In most cases the transmitted viruses establishing the infection are able to bind via the chemokine receptor CCR5 - mainly expressed on memory CD4⁺ T cells, dendritic cells and macrophages - and are called R5 viruses. Individuals with a natural CCR5 mutation are resistant to HIV-1 infection [31].

During the course of infection about 50% of the circulating clade B viruses acquire the capability to use the chemokine receptor CXCR4 as co-receptor - mainly express on naïve and memory CD4⁺ T cells; these viruses are named X4 viruses. The switch to X4 tropism is usually accompanied with a faster and more rapidly deteriorating clinical course.

A layer of matrix proteins (MA, p17) anchored to the membrane via myristolaytion forms a shell at the inner surface of the envelope [32]. MA directs the Gag (group specific antigen) precursor polyprotein to the budding site at the cellular membrane [33] and interacts with gp41 to recruit the env-encoded glycoproteins to the budding virion [34]. The mature capsid consists of about 1176 to 1356 capsid monomers (CA, p24). The structure resembles a conical fullerene lattice of around 200 hexameric units, which is closed off at both ends with 5 to 6 pentamers [35-37]. The capsid forms a protective shell for two copies of the RNA genome. The RNA associates with the nucleocapsid (NC, p7), which functions as a chaperone for the RNA. It is necessary for proper reverse transcription and protects the viral cDNA from degradation [38-40]. The small p6 protein, derived from the Gag polyprotein is essential for budding from the cellular membrane [41] and incorporates the viral protein r (Vpr) into the virus particle [42]. As part of the Gag-Pol polyprotein three viral enzymes, the protease (PR), reverse transcriptase (RT) and integrase (IN), are incorporated into the virion. Cellular lysine transfer RNA (tRNA^lys3) is bound to the RNA and serves as primer for reverse transcription.

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The genome is around 9 kilobases (kb) long and is flanked by two short repeated sequences (R) and contains a unique sequence at the 5’ end (U5) and 3’ end (U3), respectively. During reverse transcription of the RNA these elements form the long terminal repeats (LTR), that contain important regulator sequence involved in transcription initiation and polyadenylation. The four main structural proteins are encoded by gag (group specific antigen). Gag is expressed as a 55 kilodalton (kDa) precursor polyprotein (Pr55^gag), which is cleaved by the protease into matrix protein (MA), capsid (CA), nucleocapsid (NC) and p6. The main viral enzymes are expressed as a 160 kDa Gag-Pol precursor (Pr160^gag-pol), which is auto catalytically cleaved into the protease, reverse transcriptase and integrase. Both pol and gag are encoded by the same mRNA but expressed using alternative reading frames. Env is also expressed as 160 kDa precursor protein (gp160) and cleaved by cellular proteases in the Golgi into gp120 and gp41 [43, 44]. As a member of the complex retroviruses, HIV-1 encodes six additional proteins important throughout the viral replication cycle.

Three genes are expressed early in the cycle: the negative factor (Nef), trans- activator of transcription (Tat) and regulator of expression of viral proteins (Rev). Viral infectivity factor (Vif), viral protein r (Vpr) and viral protein u (Vpu) are important in infectivity, assembly and release of the virus. Vif, Vpr and Nef are present in the virion. An additional viral protein x (Vpx) is encoded by many SIV and HIV-2 but not HIV-1 or its origin virus SIVCPZ. It is also present in the virion and required in the early steps of the replication cycle [45].

1.4 Retrovirus replication cycle

1.4.1 Early steps: Membrane fusion, Reverse Transcription and Integration

As mentioned above when the gp120 molecule binds to the CD4 receptor on a target cell and a conformational change exposes the binding sites for the co-receptors.

Engagement of the co-receptors triggers an irreversible conformation change exposing the fusion peptide and the initiation of the fusion of the viral envelope and the host cell plasma membrane (reviewed here [46, 47]). Once fusion is completed the capsid core is released into the host cell cytoplasm and reverse transcription takes place (Figure 1.2). Reverse transcription is primed by the cellular tRNA^Lys3 bound to the primer binding site (PBS), 18 nucleotides in the U5 region. RT first

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synthesizes the minus strand towards the 5’ end and at the same time the RNase H activity of RT degrades the RNA in the growing RNA/DNA hybrid. When the enzyme reaches the 5’ end of the genome (R) the process comes to a stop generating a DNA fragment named “minus-strand strong-stop DNA”. Since the R sequences are identical at both ends of the genome, the R DNA stretch from the 5’ end is used to prime synthesis from the 3’ end in a process called “first strand transfer” (first jump).

The RT then continues minus strand synthesis and degrades the plus strand RNA.

Two regions are resistant to RNase H degradation – the polypurine tract (PPT) and the central PPT (cPPT). The resulting short RNA sequences serve as primers for plus strand DNA synthesis. The tRNA primer is degraded by RT when it reaches the end of the minus strand and a second strand transfer occurs (second jump) to the 5’

end of the minus strand and plus strand DNA synthesis can be completed. In this process a cDNA is generated with the full LTR (U3-R-U5) (reviewed here [48-50]).

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The early steps after release of the capsid into the host cell are still poorly understood. The entire capsid or part of the CA molecules dissociated from the cytoplasmic viral complex by a process called uncoating taking place in the first hour after fusion is completed. Not much is known about this process but the stability of the capsid core is tightly linked to proper reverse transcription. Delay of reverse transcription by RT inhibitors can alter uncoating kinetics and mutations in the CA changing capsid stability lead to defects in reverse transcription [51-53]. Several cellular factors most importantly TRIM5α and Cyclophillin A (CypA) are known to influence core stability [51, 54, 55]. The fact that HIV-1 and other viruses from the genus lentivirus including SIV can infect non-dividing cells such as terminally differentiated macrophages or resting T cells shows that HIV-1 is able to actively penetrate the nucleus of target cells. This is in contrast to gammaretorviruses such as murine leukemia virus (MLV), which rely on a disassembled nucleus during cell division for integration [56-58].

Once the pre-integration complex (PIC) arrives in the nucleus the cDNA is integrated into the host cell genome via the integrase. The integrase creates a dinucleotide CA overhang at the 3’ ends of the HIV-1 cDNA, which is covalently attached to the host genome in a process termed strand transfer reaction ([59, 60]

and reviewed here [61]). The gap occurring in the host chromosome is repaired by cellular enzymes. Another outcome of nuclear import of the PIC is the dead-end process mediated by host cell nuclear ligases where either one LTR (1-LTR circles) or two LTR (2-LTR circles) cDNA molecules are circularized [62]. These circles can be detected by PCR and are exploited as a marker for nuclear import [63]. The selection of the integration site is not completely random and is associated with markers of active transcription [64, 65]. The HIV integrase associates with the cellular chromatin tethering factor LEDGF/p75 to find the preferred integration sites [66].

1.4.2 Late steps: Viral gene expression, capsid assembly, maturation and release

Once the reverse transcribed genome is integrated into the host chromosome it is called a provirus. The LTR sequence now serves as promoter for expression of viral mRNA and genomic RNA. The 5’ LTR contains an RNA Pol II TATA box and binding sites for the transcription factor nuclear factor of activated T cells (NFAT), nuclear factor-κB (NF-κB) and activator protein 1 (AP-1). The nature of the main transcription

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factors link HIV-1 expression to T cell activation, which is necessary to activate NFAT, NF-κB and AP-1. The processivity of the RNA Pol II transcribing the HIV-1 genome is quite low and the presence of Tat enhances the HIV-1 expression. Tat binds to the trans-activation response (TAR) element, a secondary stem loop structure in the nascent HIV-1 mRNA, which leads to fully transcribed HIV-1 mRNA [67].

To express multiple proteins from a single promoter HIV-1 uses a complicated splicing scheme. There are three general mRNA products: unspliced full length genomic RNA (9 kb), single spliced mRNA (4 kb) and fully spliced mRNA species (2.2 kb). Genomic RNA encode Gag and the Gag-Pol precursor, the single spliced mRNA code for the Env precursor protein, Vpu, Vif and Vpr while the multiply spliced mRNA code for Tat, Rev and Nef [68]. Using deep sequencing methods the most recent count of HIV-1 mRNA was found to be at least 109 different spliced mRNA variants [69].

Since cellular immature and unspliced mRNA is normally retained in the nucleus, HIV-1 has a mechanism to allow unspliced mRNA export. Rev is used to achieve that goal [70-72]. Rev contains a nuclear localization signal (NLS) and nuclear export signal (NES) and can thus shuttle between the nucleus and the cytoplasm. It binds the Rev responsive element (RRE) present in all unspliced and single spliced mRNA but not in the fully spliced mRNA coding for Tat, Rev and Nef. Rev uses cellular factors to transport mRNA species with the RRE present into the cytoplasm (reviewed here [73]). Rev also serves as a switch between expression of the regulator genes Tat, Nef, Rev, the accessory proteins and Env as well as Gag-Pol.

Once enough Rev accumulates, unspliced and single spliced mRNA is preferentially transported into the cytoplasm and the proteins for assembly of new viral particles are synthesized.

Once the Gag-pol open reading frame (ORF) is expressed, the MA is immediately localized to the plasma membrane [33] and recruits Gag-Pol and NC, which in turn mediates encapsidation of the viral genome via the packaging signal (ψ) [74, 75]. NC interacts specifically with dimerized RNA genomes, which is mediated by the dimer initiation site (DIS) and required for proper packaging of the genome [76]. Env is synthesized to some extent into the endoplasmatic reticulum (ER) and transported

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via the Golgi-apparatus – where it gets glycosylated – to the plasma membrane.

Once at the plasma membrane, the long cytoplasmic tail of the gp41 interacts with MA and forms “raft”-like domains. To bud off the host membrane HIV-1 recruits the cellular ESCRT pathway [77]. Once the immature viral particle is released from the host cell the PR cleaves the Pr55^gag and Pr160^gag-pol to generate the mature and infectious particle (reviewed here [78]).

1.5 The course of HIV-1 infection

1.5.1 HIV-1 transmission and establishing infection

Different routes of transmission come with a different rate of a successful HIV-1 infection. Heterosexual transmission accounts for roughly 70% of HIV-1 infections.

The risk for a man to be infected by having intercourse with an infected woman is 1 in 700 to 1 in 3000, while it is more likely for a women to get infected by having intercourse with an infected male (1 in 200 to 1 on 2000). The remaining infections are accounted for mostly by men who have sex with men (MSM), maternal-infant infections and injection drug use. The risk of rectal transmission is considerable higher with 1 in 20 to 1 in 300. While the contact with infected blood during birth bears a 1 in 10 to 1 in 20 risk, injection drug use or accidents with HIV-1 positive blood can lead to transmission in 1 in 150 events [79]. Drug injectors and MSM have 22-fold and 34-fold higher risks to be infected with HIV-1, respectively (UNAIDS Report 2012). The lowered risk for heterosexual transmission can be attributed to the fact that the genital mucosa serves as a first physical barrier to the virus and creates a bottleneck for the initial transmitted viral population (Figure 1.3).

In fact sequence analysis and mathematical modeling of viral genomes of patients showed that in most cases only one virus/genome is establishing the systemic infection [80]. The virus crosses the genital epithelium in about 30 to 60 minutes as demonstrated in an SIV macaque model [81]. It is not clear if the virus passes the epithelial layer through the cells via endocytosis and subsequent exocytosis with or without productive infection or traverses through gaps in the epithelium and mechanical micro-abrasions in the layer occurring during intercourse. It has been shown in mice, macaques and human studies that - next to cell free virus - donor leukocyte associated virus can be the source of infection ([82-84] and reviewed here [79, 85]).

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The mucosa is lined with tissue specific dendritic cells, Langerhans cells (LCs), which belong to the subtype of conventional dendritic cells (cDC), their dendrites can extend through the epithelial layer and capture viral particle [86, 87]. The LCs express the necessary receptors for HIV-1 infection including CD4 and CCR5 but they lack DC-specific ICAM3 (intercellular adhesion molecule)-grapping molecule DC-SIGN (CD209) [88]. It is not clear if the LCs are productively infected or are just used by HIV-1 to be transferred to CD4⁺ T cells. Another cDC subtype present in the stroma below the genital epithelium, stromal DCs, on the other hand express all the necessary HIV-1 receptors including DC-SIGN, a C-type lectin receptor binding mannose rich glycoprotein present in pathogen associated patterns (PAMP) on bacteria, fungi and viruses used to sample pathogens and signal to T cells [89]. It has been shown that DC-SIGN binds gp120 and allows uptake of HIV-1 and can lead to the transfer to susceptible CD4⁺ T cells without productive infection of the DC [90].

Memory CD4⁺ T cells expressing high levels of CCR5 are present in the entire epithelium and can be infected directly by HIV-1 or receive virus via contact with LCs or stromal DCs [91].

The role of tissue macrophages in the establishment of the early infection is not clear. CCR5 expressing HIV-1 infected macrophages have been reported in cervical explants studies [92]. Furthermore macrophages are able to uptake HIV-1 particles via macropinocytosis and store the particles for several days before transferring it to susceptible CD4⁺ T cells [93, 94]. Although the extent to which DCs are productively infected in the mucosa and contribute to the initial viral replication is not clear, it has been know that the presence of DCs greatly enhance the replication of HIV-1 in CD4⁺ T cells [95]. This enhancement is due to two mechanisms. DCs are able to activate T cells via MHC class II – T cell receptor (TCR)/CD4 cross talk. T cell activation requires activated (mature) DCs, which have detected an ongoing infection (discussed below). Once the T cells are activated, the presence of NFAT and NF-κB will drive HIV-1 expression as discussed above. The second way DCs enhance HIV- 1 replication in CD4⁺ T cells is by increasing de novo infection. As mentioned above this can happen in trans via captured virus either on the cell surface or in cytoplasmic vesicles or by transferring newly produced viral particle after infection. The DCs and CD4⁺ T cells created a very close contact called the virological synapse where virus can easily be transferred to the other cell [96, 97].

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In the first 7 to 14 days the virus replicates and spreads from the initial site of infection to draining lymph nodes. This phase is called eclipse and no viremia or immune response is detected at this point [98]. Viral RNA is only detectable in peripheral blood mononuclear cells (PBMC) after 7 to 21 days and can be detected by PCR. This starts the phase of acute infection (reviewed here [79]).

1.5.2 Acute infection, latency and progression to AIDS

After the initial infection is established viremia reaches the peak with up to 10⁷ copies of viral RNA per milliliter of blood during the first 6 weeks (Figure 1.4). This phase is usually accompanied by flu-like symptoms such as fever, enlarged lymph

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nodes and joint pain. At the time of peak viremia the immune response starts to take effect and HIV-1 specific CD8⁺ T cells and neutralizing antibodies appear. This and the exhaustion of activated CD4⁺ T cells as available host cells lead to a decline in viremia of 100-fold to 1 to 10⁵ copies per ml of blood and the virus enters the latency phase.

During this time the virus replicates on a lower level and the T cell count is steadily declining. Patients can be infected from 1 to 20 years without showing any clinically symptoms until the CD4⁺ T cell count drops from initially 1000 cells per micro liter blood to fewer than 500 to 200 cells and the immune system cannot function properly any longer. At that point opportunistic infections increase leading eventually to the death of the infected patient. If left untreated HIV-1 has a mortality rate of greater than 95%. The viral load is correlated to the speed of progression to AIDS, meaning that the more virus is in the system the faster T cells are depleted and the immune system stops functioning [99]. The virus replication is supported by mostly activated CD4⁺ T cells in the blood and the mucosa. 40% of the lymphocytes reside in the gut associated lymphoid tissue (GALT), which makes up the largest part of the immune system. HIV-1 replication leads to disruption of the intestinal mucosa and destruction of the intestinal epithelial barrier and the leakage of gut bacteria into the patient’s system causing additional inflammation (reviewed here [100]). The virus levels and the rate of infected cells in the blood remain in a stable balance during the latent phase. This means that exhausted cells are getting depleted at the same rate as newly produced cells are getting freshly infected and are supporting the virus population. HIV-1 has an average replication cycle of 1 to 2 days generating more than 300 generations of genomes per year in an infected individual, providing the basis for viral evolution (quasi species). This goes on unless the patient receives the highly active antiretroviral therapy (HAART), consisting of a combination of HIV-1 inhibitors. After the first two weeks of HAART, the viremia drops 10 to 100-fold and virus levels drop below detection limit after 8 to 10 weeks of HAART (reviewed in [101]). More sensitive methods revealed that there is still virus replication ongoing in patients with full therapy and that HAART alone is not enough to remove the virus completely from the system because of the existence of a small fraction of virus producing cells of unknown origin. HIV-1 also takes advantage of the immune system to stay latent in quiescent CD4⁺T cells. Activated infected CD4⁺ T cells will go

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through rapid expansion and will give rise to effector CD4⁺ T cells. Most of them die quickly but a small fraction will differentiate into memory CD4⁺ T cells and enter a resting state, where the provirus remains in the genome but no viral gene expression takes place until the T cell is activated again [102].

The role of latently infected macrophages is still debated but they might play an important role as a reservoir, especially since infected monocytes are able to cross the blood brain barrier and differentiate into microglia cells, where they reside as tissue specific macrophages ([103-105] and reviewed here [106]). Recently it has been shown that HIV-1 can infect CD34⁺ hematopoietic stem cells (HSC) and they might serve as a long term reservoirs [107]. So far all attempts to re-activate HIV-1 from its reservoirs by CD4⁺ T cell activation with CD3 antibodies or IL2 administration combined with anti retroviral drugs has not proven to be useful to eradicate HIV-1 from infected patients [108].

1.6 The intrinsic and innate host defense

As mentioned above the host immune system is able to mount an adaptive immune response targeting HIV-1 replication using neutralizing antibodies and

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cytotoxic CD8⁺ T cells (CTLs) ([109] and reviewed here [110]). The immune system is able to control the virus for a certain period of time but eventually the capacity of adaptation will allow the virus to escape the immune response. In fact single genome analysis revealed that the virus escapes the CTL response in about 30 days after acute infection by rapidly mutating epitopes [111]. The adaptive immune response only begins to effect viral replication rate several days after the infection (reviewed here [112]).

Even before the adaptive immune system is able to respond to a threat, there are earlier defenses against pathogens. On one hand there are so-called intrinsic resistance factors. These factors are usually linked to the tropism of a pathogen, which is adapted to invade a certain cellular and molecular environment and the lack or the presence of factors can thwart the infection. For example, the absence of CCR5 in certain individuals makes them resistant to HIV-1 infection [31] or the presence of SAMHD1 inhibits reverse transcription in myeloid cells (discussed later).

In many cases intrinsic factors are linked to the innate immune system by serving not only as restriction factors but also signaling to the innate immune system that a threat is present as is the case for TRIM5 [113] or they are induced by the innate immune system (discussed later). The innate immune system is the next barrier HIV-1 has to overcome to establish an infection. It has become more and more evident that the innate and intrinsic immune systems are important in the defense against HIV-1 but might also be responsible for certain immune pathologies leading to the development of AIDS [114]. Dendritic cells are a key player in the initial steps of infection, acting as sentinels and professional antigen presenting cells (APC) in the cross talk with CD4⁺ T cells and the later CD8⁺ T cell response as well as a gateway allowing HIV-1 infection either by direct infection or as a Trojan horse transferring the virus to the CD4⁺ T cell.

1.6.1 The type I interferon system

Unlike the adaptive immune response, which has a broad repertoire of specific receptors (potentially 10⁷ to 10⁹ antigenic determinants can be discriminated) to recognize pathogens and acquire memory to a specific antigen, the innate immune system is equipped with a set of preset receptors specialized to detect non-self or danger signals - by-products of microbial replication (reviewed here [115, 116]).

These so called pattern recognition receptors (PRR) are encoded in the germline and

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are able to detect pathogen or danger associated molecular patterns (PAMPs or DAMPs) without prior encounter. The expression pattern of PRRs can be divers from just a few cell types to a wide range of cell types and tissues. Once the respective PAMP is encountered by the PRR, a signaling cascade induces the expression of interferons (IFNs), these in turn induce a broad range of interferon induced genes (ISGs) exerting diverse antiviral activities, in the producer cell itself, and in the surrounding cells. Since the receptors are already expressed in sentinel cells like dendritic cells and macrophages the response is very quick and an antiviral state can be established within minutes to hours [117]. The adaptive immune response on the other hand requires first activation signals from the innate immune system and takes days to weeks to establish a specific response towards a pathogen. The activity of IFN not only targets the replication of many pathogens but has also an effect on cellular function such as cell cycle arrest or apoptosis and can have a devastating effect on the organism (e.g. cytokine storm). Therefore the induction of the IFN system must be tightly regulated.

Interferons are grouped into three classes called type I, II and III IFNs according to their amino acid sequence. Type I IFNs consists of a large group of genes divided into IFNα (13 genes in humans and 14 genes in mice have been found) and one IFNβ gene, which are induced by viral infection. Other members of the type I IFN such as IFNε, IFNτ or IFNω have a less well defined role in other physiological pathways. IFNτ has been shown to be a multifunctional cytokine secreted by the trophoectoderm of the ruminant conceptus that manifests antiviral activity against HIV, FIV and ovine lentiviruses [118-120]. Type II IFN consists of one member called IFNγ, which is secreted by activated CD4⁺ T cells and natural killer cells (NK) rather than through viral infection. Type III IFNs called IFN-λ have been described more recently and are also induced by viral infection and activate the same pathways as IFN-α/β (reviewed here [121]).

Type I IFNs are induced by the activation of different PRRs, which are expressed in the cytoplasm, in the endosome or at the cell surface depending on their PAMP specificity (Figure 1.5). The PRRs use different downstream signaling pathways, which lead to the activation of type I IFN and proinflammatory cytokines. PRRs can be classified by their localization and the PAMP they recognize. Transmembrane PRRs of the Toll like receptor family (TLR) recognize PAMPs in the extracellular

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space (TLR2, 4, 5 and 6) and in the endosome (TLR3, 7, 8, 9). Extracellular PAMPs include mostly products from bacteria, parasites and fungi. TLR4 for example recognizes lipopolysaccharide (LPS) a cell wall component of gram-negative bacteria. TLRs of the endosome are able to detect nucleic acid: TLR 9 binds CpG DNA from viral or bacterial origin, TLR3 binds double stranded RNA (dsRNA) [122], while TLR7 and 8 bind ssRNA from viral origin [123-125].

Intracellular dsRNA is sensed by two helicases: retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (MDA5) [126]. The presence of DNA in the cytoplasm is usually a sign for viral infection or perceived as a danger signal and sensors for cytoplasmic DNA exist but the nature of these receptors and their ligand is still not well understood (reviewed here [127]). One way to sense cytoplasmic DNA is via transcription of dsDNA into dsRNA via cytoplasmic RNA polymerase III activating type I IFN using the RIG-I pathway [128, 129]. Candidates for dsDNA sensors are DNA-dependent activator of IRF (DAI) [130], DExD/H box protein DDX41 [131] and IFN inducible protein 16 (IFI16) [132]. Recently it has been shown that an enzyme called cyclic GMP-AMP synthase (cGAS) can produce a ribonucleotide moiety upon detection of cytoplasmic DNA activating type I IFNs [133, 134]. Reverse transcribed HIV-1, SIV or MLV cDNA is sensed by cGAS and induces IFNβ [135]. PRRs induce the expression of type I IFNs in a similar way using a variety of cellular signal transducer and adaptor proteins. Upon binding of dsRNA or its synthetic counterpart polyinosinic:polycytidylic acid (poly(I:C)) RIG-I and MDA5 recruit and activate a mitochondrion-associated adaptor called CARD adaptor inducing IFN-β (Cardif)/virus-induced signaling protein (VISA)/mitochondrial antiviral protein (MAVS)/IFN-β promoter stimulator protein 1 (IPS-1). MAVS interacts with tumor necrosis factor (TNF) receptor-associated factor 3 and 6 (TRAF3, 6). The E3 K63 ubiquitin ligase activity of TRAF6 will lead to autoubiquitination recruiting TAK1 binding proteins 2 and 3 (TAB2, 3), which serve as a platform to assemble the signaling cascade complex including transforming growth factor β-activated kinase 1 (TAK1). NF-κB is retained in the cytoplasm via inhibitor of NF-κB (IκB) and TAK1 phosphorylates the kinase of IκB (IKKα/β). IKKα/β then releases NF-κB from IκB leading to the translocation of NF-κB into the nucleus. On the other arm of the pathway TRAF3 binds directly to TRAF family member associated NF-κB activator (TANK), which in turn recruits TANK-binding kinase-1 (TBK-1). TBK-1

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phosphorylates the transcription factor IFN regulatory factor 3 (IRF3) and to some extent also IRF7. Phosphorylation of IRF3 leads to dimerization and exposes a nuclear localization signal (NLS). The IRF3 dimer then translocates into the nucleus where, together with IRF7, NF-κB, CBP/p300 and RNA polymerase, it activates expression of the IFNβ gene and other proinflammatory cytokines. The DNA sensors DDX41, IFI16 and cGAS activate TBK-1 via the ER associated adaptor called stimulator of IFN genes (STING) inducing IFNβ expression via IRF3 [136]. TLR3 and TLR4 signaling activates IRF3 and NF-κB directly via an adaptor called Toll-IL1 resistance (TIR) domain-containing adaptor inducing IFNβ (TRIF) and subsequent TBK-1 activation without the involvement of MAVS. Additionally, TLR3 and TLR4 activated AP-1 via the mitogen-activated protein kinase (MAP) pathway. NF-κB modulated by AP-1 induces the expression of proinflammatory cytokines like tumor necrosis factor α (TNFα), Interleukin 1 (IL1) and IL6 being the most important ones.

TLR7, 8 and 9 on the other hand recruit myeloid differentiation factor 88 (MyD88), which then recruits TRAF3 leading to IRF7 phosphorylation and TRAF6 leading to NF-κB activation via TAK1 inducing IFNβ without the involvement of IRF3.

Type I IFNs act on the secreting cell itself (autocrine) as well as on neighboring cells (paracrine). IFNα/β bind to a heterodimeric receptor composed of IFNAR1 and IFNAR2 gene products (IFNαβ receptors 1 and 2). The cytoplasmic tail of IFNAR1 is associated with tyrosine kinase 2 (Tyk2) and the cytoplasmic tail of IFNAR2 is associated with the tyrosine kinase Janus kinase 1 (JAK1). Upon ligand induced dimerization, Tyk2 phosphorylates IFNAR1, creating a docking site for signal transducer and activation of transcription 2 (STAT2) and a weak association with STAT1. STAT2 is then phosphorylated by Tyk2, while JAK1 phosphorylates STAT1.

That permits the dimerization of STAT1 and STAT2 which are then transported into the nucleus, where this heterodimer recruits IRF-9, forming the heterotrimeric IFN stimulated gene factor 3 (ISGF3). ISGF3 binds to the IFN-stimulated response element (ISRE), which is present in the promoter of most ISGs and initiates their transcription. Several hundred genes are known to be induced by IFN signaling, such as the protein kinase R (PKR), the 2’5’-oligoadenlyte synthetase (OAS)/RNaseL system, Mx proteins and proteins of the APOBEC family and TRIM family (discussed below). PKR uses dsRNA as cofactor and phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), which interrupts mRNA translation.

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The OAS/RNaseL system degrades cellular and viral RNAs. Mx proteins are small dynamin-like GTPases, which exhibit antiviral activities against a wide range of RNA viruses including Influenza [137, 138].

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Although HIV-1 is very sensitive to the action of type I IFNs and its replication can be inhibited in cells in an antiviral state 100 to 1000-fold [139], in patients type I IFNs are not able to control the virus since using it as a treatment failed to give positive results. In fact it may actually be detrimental for the patients and might contribute to progression to AIDS (reviewed here [140]). Our knowledge of how HIV-1 is sensed by the innate immune system and induces IFN or avoids detection is still limited but in recent years the picture became clearer. As most viruses HIV-1 has evolved strategies to avoid detection by the innate immune system and evade antiviral activities (reviewed here [121, 141, 142]). The main cell type producing type I IFNs are plasmacytoid dendritic cells (pDCs). They are the only cell type next to B cells to express TLR7. Via endocytosis pDCs take up HIV-1 particles and the genomic ssRNA is sensed by TLR7 in the endosome [143]. Cell free virus is able to stimulate type I IFN induction but the stimulation is greatly enhanced when pDCs make cell to cell contact with infected CD4⁺ T cells [144]. In the same study it was shown that fusion was not required for TLR7 stimulation with cell free virus in the case of cell to cell contact fusion lead to a greater stimulation of type I IFN. The exact HIV-1 ligand for TLR7 is not known but HIV-1 ssRNA must be delivered to the endosome after fusion but before reverse transcription takes place. Probably cytosolic ssRNA is taken up by the early endosome via autophagy since knockdown of Atg7 in pDCs led to a decrease in type I IFN production after HIV-1 infection [145]. Conventional DCs but not pDCs express TLR8 and HIV-1 infected DCs showed increased replication mediated by both TRL8 signaling and DC-SIGN signaling [146]. One report shows that newly produced capsid in DCs is able to induce type I IFN via IRF3 and cycophilin A (CypA) [147]. CypA has been shown to bind very strongly to HIV-1 CA [148] and is able to promote an early step in the HIV-1 replication in specific cell types [149]. It is not clear how CypA is stimulating type I IFN production but the involvement of an unknown sensor has been postulated [147].

1.6.2 Intrinsic HIV-1 restriction factors BST-2 and Vpu

As mentioned above there are several restriction factors known to block different steps of the HIV-1 replication cycle and HIV-1 accordingly has evolved countermeasures to destroy or circumvent these factors. Analyzing the cell type

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specific requirement of Vpu for release of viral particle from the host cell membrane led to the discovery of B cell stromal factor 2 (BST-2, CD317 ). This factor is greatly induce by type I IFNs and it has been shown to be degraded by Vpu [150, 151].

Because of its function BST-2 is also called tetherin [152]. It is a highly glycosylated protein embedded into the cell membrane. HIV-1 particle are still able to bud from the membrane but BST-2 anchors the released particles to the membrane. The interaction seems to be rather unspecific since it is able to tether and inhibit other enveloped viruses such as influenza [153]. Vpu also is involved in decreasing surface CD4 expression after infection [154]. The same function is also exerted by Nef [155]. Vpu recruits CD4 to the Skp1 binding protein (β-TrCP) inducing polyubiquitination and subsequent proteasomal degradation at the ER [156, 157] and also uses the same pathway to degrade BST-2 [158]. The removal of CD4 and BST- 2 both increase the release of viral particle and are essential for HIV-1 replication.

Nef additionally downregulates MHC class I molecules [159] and the T cell receptor [160] to possibly inhibit CTL response. Interestingly, only the M group viruses achieved full adaptation to escape BST-2, which might have allowed the virus to become pandemic (reviewed here [161]).

ABOPEC3G and Vif

Similar analysis as with Vpu led to the discovery of the restriction factor degraded by Vif, apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G or shorter APOBEC3G (A3G). As the name indicates A3G deaminates cytidine to uridine introducing G to A hyper mutations during reverse transcription rendering the genome inert [162]. Indeed the fingerprint of A3G activity has been found in endogenous retroviruses [163]. A3G is incorporated into newly released viral particles by interacting with the genomic ssRNA unless it is targeted for proteasomal degradation by Vif, which recruits the Cullin5-RING finger E3 ubiquitin ligase (CUL5) [164, 165]. There is also evidence that APOBEC3G inhibits elongation of the reverse transcription by its physical presence and independently of its catalytic activity [166].

DDB1 and Vpr

The use of the Cullin-RING (really interesting new gene) finger ligases (CRL) is a common way adopted by HIV-1 to use the ubiquitin-proteasome system (UPS) to degrade cellular factors. CRLs serve as a scaffold to assemble substrate receptors to the close proximity of the E2 and E3 ubiquitin conjugating enzyme. Vpr binds to

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DDB1-and Cullin4A-associated factor 1 (DCAF1) also termed Vpr binding protein (VprBP), which in turn binds to damage-specific DNA binding protein 1 (DDB1).

DDB1 is part of the pathway to sense ultra violet light damaged DNA inducing the base excision repair (BER) machinery. DNA damage induces cell cyle arrest in G2. It is not completely understood how but Vpr is able to induce cell cycle arrest by binding to DCAF1[167-169]. Different mechanisms have been proposed; Vpr-DCAF1 binding could interfere with the recruitment of the natural substrate of DCAF1, increase the activity of the CRL complex or recruit a cellular factor for degradation.

Uracil-DNA glycosylase (UNG), an enzyme involved in the BER by removing uracil from DNA, was shown to be degraded by Vpr [170]. Vpr of HIV-1 shares 86%

homology with Vpx from SIVMAC and HIV-2 [171] and it is not surprising that Vpx uses the DCAF1-CUL4A complex to degrade its cellular target SAMHD1 (discussed below).

TRIM5α and the retroviral capsid

In 1957 Charlotte Friend observed that some mice were resistant to certain viral strains but not others and vice versa. The strains were later termed N-murine leukemia virus (NIH Swiss mice tropic) and B-MLV (BALB/c mice tropic) [172].

Through genetic experiments it was found that the restricting allele was dominant and the locus termed Friend virus susceptibility 1 (Fv1) [173, 174] and this restriction activity could be mapped to a single amino acid in the MLV CA [175]. When the gene was later cloned it turned out to be of endogenous retroviral origin but the precise mechanism of restriction for MLV is still not completely understood [176]. N-MLV is restricted in human cells and HIV-1 in monkey cells in a similar fashion as MLV in murine cells and the activities were termed resistance factor 1 (Ref1) and lentivirus susceptibility factor 1 (Lv1) in accordance to Fv1. In 2004 two groups were able to identify the genes of these loci, they cloned TRIM5α from rhesus macaque cell cDNA library [177] and TRIMCyp from a Owl monkey cell cDNA library [178]. The TRIM5 from Owl monkey turned out to be a fusion with TRIM5 and CypA created by a LINE1 retrotransposition event [178]. Interestingly, a similar independent fusion event was also found in certain macaque species [179-182]. An engineered fusion of human TRIM5 and human CypA showed potent HIV-1 restriction in both human CD4⁺ T cells and macrophages and in a humanized mouse model [183]. TRIM5α is a member of the tripartite motif-containing (RBCC) gene family and consists like most of the TRIM

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proteins of a RING finger domain (R), B-box domain (B), coiled coil domain (CC) and in the case of TRIM5α a PRYSPRY domain (or B30.2) (Figure 1.6). The PRYSPRY domain is responsible for HIV-1 capsid binding and restriction [55, 184] and although TRIM5α restricts HIV-1 lab strains only weakly (around 2-fold) it can restrict certain primary isolates up to 10-fold [185]. TRIM5α only binds and restricts the fully matured capsid but not newly synthesized CA protein. The exact binding of TRIM5α on the surface of the capsid cone has not been determined yet but it probably interacts with the inter-hexameric trimer interface [186]. The RING finger domain is an active E3 ubiquitin ligase [187] and is required for restriction [113, 188]. The B-box domain and coiled-coil domain are both required for TRIM5α multimerization [187, 189]. TRIM5α probably binds the incoming capsid resulting in premature uncoating through an unknown mechanism before reverse transcription is completed (reviewed here [141, 142, 190, 191]).

1.6.2.1 The role of Aicardi-Goutières Syndrome genes in HIV-1 replication In 1984 Jean Aicardi and Françoise Goutières first defined Aicardi-Gouitères Syndrome (AGS) as a genetic disorder in new born and young children [192] showing symptoms that resemble a congenital encephalitis caused by infection with Toxoplasma, HIV-1, CMV or Rubella (TORCH) including high levels of IFNα in the cerebrospinal fluid (CSF) and brain calcifications leading to an early progressive encephalopathy (reviewed here [193, 194]). Mutations in six different genes are associated with AGS: TREX1 [195], the three subunits of the RNase H2 complex (A,B and C) [196], SAMHD1 [197] and ADAR1 [198] (all discussed below). The mechanisms behind the disease have not been fully understood. It has been postulated that a defect in nucleic acid metabolism, more specifically accumulation of microRNAs [199], cytoplasmic single stranded DNA from endogenous

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retroelements [200] or DNA repair byproducts [201] lead to the activation of innate immune receptors such as RIG-I or DNA sensors and the subsequent up regulation of type I IFNs and other inflammatory cytokines. Surprisingly, all of the genes have been found to play a role in HIV-1 replication and innate immune sensing either restricting or facilitating HIV-1 infection (Figure 1.7).

ADAR1

Adenosine deaminase acting on RNA 1 (ADAR1) belongs to a class of enzymes that edit mRNA. The deaminated adenosine becomes an inosine and pairs with cytosine instead of thymidine introducing A to G mutations much like A3G. RNA editing changes amino acid sequences [202], splice sites [203] and introduces new miRNA binding sites [204]. ADARs have been found to be involved in innate immunity by hyper-mutating genomes of RNA viruses such as Measles [205] and vesicular stomatitis virus (VSV) and also mouse polyoma DNA virus [206, 207].

ADAR1 expression is induced by type I IFNs and it has been found to be able to modify the innate immune response by sequestering dsRNA away from PKR inhibiting the activation of PKR and acting as a suppressor of type I IFN signaling ([208, 209] and reviewed here [210, 211]). Recently it has been reported that ADAR1 mutated the HIV-1 RRE, inhibiting Rev dependent nuclear export of gag and pol transcripts and inducing mutations in Env rendering the produced particle less infectious [212]. ADAR1 knockout mice develop sever deficits in hematopoiesis and showed global upregulation of type I and type II interferon [213].

HIV-1 innate immune detection and evasion

Thesis

Reference

HIV-1 innate immune detection and evasion

HIV-1 Innate Immune Detection And Evasion

THESE

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

UNIVERSITE DE GENEVE

Docloro] ds sciences Mention biologie

Thdse ae lllonsieur Christian REINHARD

" HIV-I lnnofe lmmune Deleclion And Evosion "

Thdse - 461 0 -

Doyen,

“Non est ad astra mollis e terris via“

Table of contents

Abstract

Résumé

List of Abbreviations

Chapter 1: General Introduction

1.1 Three decades of HIV/AIDS

1.2 The origin of the HIV pandemic

1.3 Virion structure and genomic organization of HIV

1.4 Retrovirus replication cycle

1.5 The course of HIV-1 infection

1.6 The intrinsic and innate host defense