Figure 5: Structures of the HIV-‐1 genome and virion. A) The HIV-‐1 genome is composed of two flanking LTRs containing typcial retroviral regulatory sequences, and different ORFs coding for gag-‐pol, env, the regulatory proteins rev and tat, and the accessory proteins vif, vpr, vpu and nef. The rev-‐responsive element (RRE, light blue box) is depicted below the env ORF (green box). B) The structure of the HIV-‐1 mature virion is depicted with the cleaved viral products, which are indicated with arrows. A) and B) from Sakuma et al, 2012 80.
1.1.8 The HIV-‐1 life cycle
The first step of the HIV-‐1 replication cycle is the viral particle fusion with the plasma membrane of a susceptible cell that expresses specific receptors. Upon the binding of gp120 to the CD4 receptor of an immune cell such as a
lymphocyte, it further interacts with a specific seven transmembrane domain G protein-‐coupled coreceptor depending on the virus tropism. Generally, CCR5-‐
tropic virus dominate the first phase of the infection, and gradually the tropism of some strains can change to reach up to 50% of viral particles that use the alternative CXCR4 co-‐receptor 81,82. Additionally, some strains of HIV-‐1 are able to use other coreceptors as CCR2b, CCR3, CCR8 and the orphan receptors V28, STRL33 and GPR15 83.
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Once the virus fuses with the membrane, the viral core is released into the cytoplasm, where reverse transcription takes place. It is still a matter of debate where the capsid uncoating may occur, before reverse-‐transcription, concomitantly or after completion of the process. Three major models are postulated, as reviewed in 84. One of them put forward the hypothesis that the total uncoating is necessary to activate the reverse-‐transcription complex (RTC).
This first hypothesis was based on the observations that low-‐levels of capsids were recovered from HIV-‐1 complexes extracted from cells and on the inability to visualize the capsid complexes with Transmission Electron Microscopy (TEM) within infected cells 85-‐87. These findings were not able to discriminate between partial and total uncoating.
Based on the results from a study showing that CA associates with the RTC, two other schools of thoughts emerged 88. The first alternative model implies that the capsid uncoating happens gradually, upon environmental changes encountered by the viral core, like the interaction with different cellular factors at different steps of the reverse-‐transcription process and the production of cDNA and reverse-‐transcription intermediates. This hypothesis is reinforced from one side, by the observation of different sizes of HIV-‐1 cores isolated from early infected cells and that diverge from the mature HIV-‐1 complexes 88,89. From another side, the findings that altering the stability of the capsid core via mutagenesis of specific CA residues to either an increase or a decrease will affect the completion of the reverse-‐transcription are in agreement with this model 90. Whereas the first observation could simply represent an artifact coming from the biochemical method used for the virus isolation, the second finding uses mutations that could induce conformational changes that modify the interactions with host factors and the RTC in in vivo experiments that are not relevant in the course of a natural infection. Nevertheless, TRIM5, a factor that will be described later, blocks retroviruses at an early infection step, via accelerated CA core disassembly. This observation reinforces the model in which a tightly controlled uncoating is necessary for an efficient viral replication.
The second alternative model postulates that the capsid remains associated with the RTC until completion of the reverse-‐transcription at the nuclear pore. The finding that the capsid is required for proper nuclear import 84,91 argues in favor of this model, but does not contradict the postulate where uncoating is a gradual event.
Alternatively, the uncoating could possibly not occur, as shown by the experiments done by Burdick and colleagues 92.
Nevertheless, the CA uncoating of at least a portion of particles seems to happen very early after viral entry, as shown by experiments using the CA-‐specific blockade of HIV-‐1 by TRIM5Cyp, which will be described later. TRIM5Cyp-‐
mediated restriction is mediated by recognition of intact cores and this binding is impeded by Cyclosporine A (CsA), as refered below. Taking out the CsA treatment (CsA washout assay) after 2 hours precludes definitively restriction, indicating that the CA stability was disrupted before this time point 93. Similarly, immunofluorescence microscopy allowed detecting less particles associated with capsid after 1 hour. In the same study, the chemical inhibition of the RT, using nevirapine (NVP), in combination with the CsA washout assay showed that the reverse-‐transcription progression is necessary for normal uncoating, as this process did not become apparent when the inbitory drug was used 93. The restriction factor TRIM5, recognizes the capsid hexameric lattice. It is therefore possible that even if only partial uncoating happen, TRIM5Cyp wouldn’t bind to the particle anymore. Further evidence that uncoating is a rapidly started event comes from a study where another set of restriction factors, the APOBEC3 proteins, act on the nascent cDNAs, in the target cell 94. This finding suggests that the RTC is accessible very early within the viral core to proteins present in the cytoplasm, before or at the time of reverse-‐transcription 95.
Concomitantly, there are several results suggesting that CA remains associated until a late step of retroviral replication. First, experiments show that CA is the determinant for nuclear import 71,96,97. Second, CA interacts with Cyclophilin A (CypA) and this interaction is required for proper reverse-‐transcription but blocks nuclear-‐entry in some cell lines 98. Third, a study revealed that CA binds to
the CypA domain of the Nucleoporin protein of 358 kDa (Nup358) at the nuclear pore complex. Interaction with this protein allows nuclear entry of the PIC 99. When Nup358 or another member of the complex involved in this nuclear internalization pathway -‐ TNPO3 or cytoplasmic CypA-‐ are disrupted, other routes are used and result in different preferential integration sites and concomitant impaired HIV-‐1 replication, as reviewed by Fassati 95. Fourth, CA binds to nuclear export factors, suggesting it can localize to the nucleus 100. Fifth, CA total uncoating is not necessary for reverse-‐transcription to proceed, as a mutant that stabilizes the CA core still synthesizes normal levels of cDNA 93.
The observation that different natural or artificial restriction factors recognizing the CA block different pre-‐integration steps, including post-‐nuclear entry, further argues in favor of a partial and gradual uncoating that culminates into the nucleus at some step before integration 101,102 103. Despite a considerable body of work devoted to the HIV-‐1 uncoating process, the subcellular compartment and the viral replication step where its completion occurs remain a mystery. What is clear from previous studies is that a proper timing of CA uncoating is necessary for various steps of the viral life cycle to proceed, as shown by the CA-‐dependent negative effect on HIV-‐1 replication by restriction factors that accelerate the uncoating (see below) and by proteins that are influencing nuclear entry.
The process of the nuclear import of the PIC seems to rely on the capsid protein.
In earlier studies, IN, MA and Vpr were suggested to be required for nuclear entry and the fact that these proteins carry a nuclear localization signal (NLS) supported this theory. To understand the key experiments that were performed to investigate the viral proteins involved in nuclear import of the PIC, one must consider that there is a major difference between lentiviruses and other retroviruses like the gammaretrovirus MLV, in respect to nuclear entry.
As such, whereas HIV-‐1 can enter the nucleus of non-‐dividing cells, MLV is dependent on the breakdown of the nuclear membrane at the mitosis to import its PIC 104-‐106. This feature in fact allows the gammaretrovirus to uncoat after nuclear entry 107, perhaps conferring a protection of the RTC and the PIC from cytoplasmic sensors. Taking advantage of this difference, the IN or the CA
proteins of both retroviruses were exchanged, expecting that they could confer a differential ability to enter the nucleus.
When adding a NLS into the MLV MA or IN proteins, MLV could still not be imported into the nucleus of resting cells 108,109. Interestingly, however, the exchange of the MLV capsid by the one from HIV-‐1, transferred to the gammaretrovirus the independency from cell-‐cycle requirements for nuclear entry 91. Reciprocally, HIV-‐1 with an MLV capsid lost its ability to infect non-‐
dividing cells.
To import the PIC into the nucleus, nuclear pore complex (NPC) proteins as Nup98, Nup153 and Nup358 and other cellular factors seems to be required, as reviewed by Fassati 95. Before nuclear entry, the PIC machinery already activates the viral DNA to be integrated 69. IN binds to both viral LTRs, recognizing internal specific sequences and catalyzes the processing at a CA dinucleotide, producing an available 3’ hydroxyl group, which constitute a cleaved donor complex (CDC) that is competent for nucleophilic attack of the target DNA once in the nucleus. This interaction leads to the DNA strand transfer in whom the 3’
ends of the viral cDNA are ligated to 5’ phosphates into the host chromatin, as reviewed by Krishnan and Engelman 69. Once the strand transfer complex is formed, repair enzymes from the host take care of joining and filling the gaps created at the 5’ ends of viral cDNA within the targeted host genome, creating the so called target site duplication (TSD) at both ends of the provirus 69.
As reviewed by Karn and Stolzfus 78, regulation of HIV-‐1 expression from the provirus is controlled both transcriptionally and post-‐transcriptionally. The two accessory proteins tat and rev are responsible for the stimulation of the transcripts elongation and the export of some mRNAs species, respectively, that would otherwise be degraded within the nucleus. Within HIV-‐1 LTR, the transactivation-‐responsive element (TAR) recruits tat and its cellular cofactor P-‐
TEFb, resulting in transcriptional elongation, a process that is dependent on cellular elongation factors as ELL2 78.
The core promoter of HIV-‐1 contains three SP1 binding sites, a TATA box and an initiator sequence. Additionally, the HIV-‐1 LTR bears an NFKB binding site, which acts a viral enhancer involved in the reactivation of latency and in increased HIV-‐1 replication in T cells 78,110. The epigenetic regulation, dependent on acetylation and methylation of histones as well as on DNA methylation allows the virus to establish latency 111. At a later stage, HIV-‐1 transcripts are exported from the nucleus to the cytoplasm. Given that unspliced and incompletely spliced mRNAs are the target of nuclear enzymes that degrade them, these transcripts species have to associate with rev, that recognize a specific sequence in the env coding sequence, the rev-‐responsive element (RRE) and hide them from the cellular machinery. The protected transcripts are then exported through the NPC via interaction with the cellular protein Crm1 78.
HIV-‐1 transcripts are further processed at the 3’ end and polyadenylated, in the view of being translated together with host-‐derived mRNAs 78.
The gag gene products that constitute the structural components of the HIV-‐1 virion coordinate the last phase of viral replication. Indeed, viral proteins and nucleic acid materials assembly at the viral membrane is directed by the unprocessed gag polyprotein that binds the plasma membrane, and the env protein, recruits the PR, the RT and the IN proteins and packs the viral RNA and the primer tRNALys2, 3 112, forming an immature virion. During the budding process, the plasma membrane is integrated into the viral particle, constituting a de novo lipid bilayer. Upon particle maturation, the PR protein cleaves gag, producing processed MA, CA and NC. The resulting mature virions are either released into the blood stream or directly infect new cells via cell-‐to-‐cell transmission involving the formation of virological synapses 39,113.
My thesis will focus on the activity of the restriction factor TRIM5, which will be introduced further in the next chapter, and thus I will examine the early steps of retroviral replication.
1.2 TRIM5 and the innate immunity
Viruses and other pathogens attack the organism by different routes. The immune response that is mounted to counteract this invasion depends on different germline-‐encoded and de novo synthesized factors that are produced in the context of the innate and adaptive immune response, respectively. As a first defense, all cell types that are the target of infections carry different combinations of proteins acting like sentinels that recognize specific foreign motifs or molecular signatures. These pathogen-‐associated molecular patterns (PAMPs) are bound by pattern-‐recognition receptors (PRRs), at the cell membrane, in endosomal compartments or within the cytoplasm, and this interaction results in the activation of signaling pathways that will ultimately lead to the production of inflammatory cytokines and type I Interferon (Type I IFN), as reviewed in 114The inflammatory cytokines activate immune cells and act on endothelial cells, in this way stimulating the early inflammatory response (reviewed in 115).
Secretion of Type I IFN provokes an antiviral state via the production of interferon-‐stimulated genes (ISGs) and the stimulation of the acquired immune system by activating immune cells, contributing to the presentation of Major Histocompatibility Complex Class I (MHC I) molecules, essential for recognition of antigens exposed by antigen-‐presenting cells (APCs) and promoting cytotoxic T lymphocytes (CTL) response 116.
Type I IFN stimulates the expression of a plethora of factors with different specificities to mount a broad antiviral response. The category of ISGs contains several hundreds of genes that serve as antiviral effectors or as signaling activators for processes such as apoptosis and vesicular transport 117. Many antiviral effectors are components of the intrinsic immunity, and will be discussed later. These constituvely-‐expressed factors are able to degrade viral components specifically in a direct way.
Interestingly, as it will be discussed later, some of these ISGs, including TRIM5, function as PRR them selves, showing the way by which the innate immunity can be self-‐amplified.
1.2.1 The Pattern-‐recognition receptors
Four main families of PRR are associated with detection of foreign material within a cell: one group of membrane-‐associated receptors and three classes of cytoplasmic PRRs.
Toll-‐like receptors
A first group of PRRs is composed of proteins with a transmembrane domain, an extracellular leucine rich repeat (LRR) region that recognizes specific PAMPs, and an intracellular module containing a TLR/IL-‐1R (TIR) domain that allows them to interact with adaptors molecules for the signal transduction 118.
With ten functional members in humans, the Toll-‐like receptor (TLR) family recognizes a wide variety of PAMPs. Using different adaptor molecules, TLRs bind to many types of molecules including lipids, lipoproteins, proteins and nucleic acids. The members of this family of receptors are located at different cell compartments. Whereas TLRs 1, 2, 4, 5, 6 and 10 are found at the cell surface, TLRs 3, 7, 8, and 9 are located in endosomes 114,119 (figure 6). TLR2 is found in the form of a dimer, either with TLR1, TLR6 or TLR10 mainly detecting PAMPs from Bacteria and fungi 114,120,121. When complexed with TLR1, the dimer recognizes the triacetylated lipoproteins, peptidoglycans and lipopolysaccharides 122, as reviewed by Kawai and Akira 114.
The dimer composed of TLR2 and 6 is responsible for the detection of diacylated lipoproteins 123. The specific molecules recognized by TLR2-‐TLR10 have not been discovered yet 120,121. The other cell surface-‐associated PRRs, TLR4 and 5, were found to bind to lipopolysaccharides (LPS) and flagellin 124, respectively (reviewed in 125).