1.2.3 Immunity to retroviruses: restriction factors
The replication ability of retroviruses in different cells depends on many cellular factors. The first considered factor is the entry of the retrovirus into the cell cytoplasm, via recognition of the corresponding receptor. For example, as discussed previously, HIV-‐1 entry requires the recognition of the CD4 receptor and a coreceptor, principally CXCR4 or CCR5. The subsequent steps of the viral life cycle exploit host proteins in a species-‐dependent way to proceed, as highlighted by the inability of HIV-‐1 to productively infect murine cell lines that have been engineered to express human CD4 182 and taking into account that the murine CXCR4 can be used as a coreceptor by HIV-‐1 183. Importantly, murine cells have a cyclin T1 protein, that HIV-‐1 Tat does not bind because of a species-‐
specific polymorphism, thus precluding the employment of this cofactor required for the transactivation of LTR-‐directed expression 184. When circumventing this post-‐entry blocks by expression of human Cyclin T1, some murine cell lines proceed into viral gene transcription, but further steps are blocked, as mRNA export and processing, as well as virion assembly 185,186. These blockades are rescued upon fusion of murine and human cells, showing that there are factors exerting a positive effect on viral replication late steps that are not present in the mouse 187.
Interestingly, in contrast to fibroblasts, murine T cells do not support HIV-‐1 reverse transcription 183. The blockade of a pre-‐integration step of the viral replication strongly recalls other phenotypes observed in mice and primates.
The cell tropism is not only dictated by the presence or the absence of positive cofactors in a cell.
The first indirect report of a negative factor influencing retroviral replication was in 1957 by C. Friend who discovered that a genetic transmissible trait
dictated the susceptibility of different strains of mice to MLV 188. The factor mediating this blockade was later genetically mapped on chromosome 4 and called Fv-‐1 189,190. The two alleles of the gene were Fv1B and Fv1N that conferred the resistance to N tropic MLV (N-‐MLV) and B-‐MLV, respectively. Nineteen years after Friend’s discovery, the blockade by Fv-‐1 was determined to act after reverse-‐transcription, but before integration 191 (figure 8).
In parallel with the previous findings, a potent blockade of HIV-‐1 infection was observed in monkey cells 192,193 and was termed lentiviral susceptibility factor 1 (Lv1). Few years later, an Fv-‐1B-‐like restriction was observed in cells from primates including human, and from dog, pig and cow that potently blocked N-‐
MLV early post-‐entry 194. The host protein responsible for this blockade was called restriction factor 1 (Ref1).
In common to all restriction factors, the barrier to retroviruses was capsid-‐
specific, dominant and saturable by a high amount of viral particles 195-‐199. The factors at the origin of these blockades were all cloned and it appeared that Lv1 and Ref1 were products of the same gene, TRIM5 62,200-‐203.
I will first briefly describe the best-‐studied restriction factors affecting retroviral replication and will then focus on TRIM5.
Figure 8: The blockade of early retroviral replication steps by TRIM5 and Fv1. The restriction factors TRIM5 and Fv1 inhibit retroviral replication at an early post-‐entry step. Whereas TRIM5 can act before reverse-‐transcription and nuclear import (solid black bars), Fv1 only targets the latter step. The virion core is represented by the blue conical shape. RNA and DNA species within the core are depicted as two black and blue bars, respectively. Courtesy of Prof. Jeremy Luban (adapted).
APOBEC3 proteins
Among a family of cytidine deaminases, the apolipoprotein B mRNA-‐editing enzyme catalytic polypeptide-‐like 3G (APOBEC3G) inhibits the replication of HIV-‐1 by associating with assembling virions, via its N terminal zinc-‐binding deaminase domain that interacts with the viral RNA and gag polyprotein 204,205. Once in a target cell, APOBEC3G recognizes cytosine residues within C-‐C dinucleotides on newly synthesized minus-‐strand viral cDNA and induces their deamination, transforming it into a uracil 206. The resulting viral genome contains guanine to adenine mutations, leading to replication catastrophe 207. In addition, the catalytic activity of APOBEC3G is required for the blockade of HIV-‐1 integration. The mechanism relies on the interference with the tRNALys3 primer dissociation, leading to the formation of abnormal 3’LTRs and thus a subsequent defect in its targeting to the host genome 208. Finally, APOBEC3G targets HIV-‐1 at
TRIM5 Reverse Transcription
Nuclear Import Fv1
the reverse-‐transcription step by impeding the tRNALys3 to prime the viral RNA, although it is not clear whether this is in a deaminase-‐dependent way 209-‐212. However, despite these potent restrictions, HIV-‐1 evolved a mean to counteract APOBEC3G by orchestrating its degradation by the viral accessory protein Vif in a proteasome-‐dependent pathway 206.
Other members of the APOBEC family have similar deamination activity and restrict HIV-‐1 infection 213. While APOBEC3A has been linked to the inhibition of HIV-‐1 in monocytes 214, APOBEC3B is not expressed in primary lymphoid cells but still renders HIV-‐1 particles less infectious, when expressed transiently in the virus-‐producing cells 213,215. The other APOBEC proteins exerting anti-‐HIV-‐1 activity include APOBEC3C that inhibits the infectivity of some strains of HIV-‐1
216, APOBEC3D/E that is counteracted by vif 217 and APOBEC3F 213,218,219.
Tetherin
The tetherin restriction factor, named in that way because it “tethers” HIV-‐1 virions to the cell surface, impeding their release 220. By homodimerizing via the extracellular coiled-‐coil domain, tetherin engage a second monomer bound to the viral membrane 221.
Tetherin is induced by type I IFN and its action is counteracted by HIV-‐1 Vpu 220. In turn, the activation of the NFκB pathway by tetherin upon HIV-‐1 infection 222 results in the production of type I IFN.
SAMHD1
At first associated with the Aicardi-‐Goutières autoimmunity syndrome 223 the sterile alpha motif (SAM) and histidine-‐aspartic (HD) domains-‐containing protein 1 (SAMHD1) was subsequently investigated for its role in mediating the innate immunity to retroviruses. This restriction factor was found to decrease dNTP levels and to block HIV-‐1 reverse-‐transcription 224. As for APOBEC3G and tetherin, some retroviral accessory proteins neutralize SAMHD1. Indeed, Vpx
from HIV-‐2 and SIV degrades SAMHD1 by targeting this factor to the proteasome
225. MX2
The IFN-‐induced myxovirus resistance 2 (MX2) protein restrict HIV-‐1 infection in a capsid-‐dependent way 103. Although the mechanism of retroviral inhibition remains unknown, the transient expression of the restriction factor decreased 2-‐
LTR circles formation and integration 103, suggesting that this protein inhibits HIV-‐1 nuclear entry.
ZAP
The first retroviral target of the zinc-‐finger antiviral protein (ZAP) to be discovered was MLV 226. In this study, the abundance of MLV transcripts was decreased in rodent cells expressing the endogenous protein. It was later found that HIV-‐1 was similarly restricted by the human ZAP orthologue, which induced specific mRNA uncapping and degradation of the retroviral transcripts 227.
MOV10
Discovered in murine strains as the site of MLV provirus integration 2. the MOV10 gene encodes a protein with seven helicase motives 228. The human orthologue of MOV10 inhibits HIV-‐1 at various replication steps. Although the mechanism by which MOV10 reduces HIV-‐1 virion production is still unclear, it could involve the inhibition of gag expression 229 and this could have a link with the observed association of MOV10 orthologues from mammals with the RNA interference (RNAi) system 230 that may silence viral gene expression. At a second level, virion-‐associated MOV10 from the producer cells restricts HIV-‐1 reverse-‐transcription in the target cells 229,231.
ADAR-‐1
The adenosine deaminase acting on RNA protein 1 (ADAR1) induces the deamination of adenosine into inosine on a double-‐stranded RNA substrate 232. It was recently found that this enzyme has a restriction activity on HIV-‐1, inhibiting the expression of viral proteins via the post-‐transcriptional mRNA editing inducing a defect on nuclear export of the respective messengers of gag, pol and env 233.
Fv1
The sequence of the murine restriction factor Fv1 is derived from a gag gene from the endogenous retrovirus family ERV-‐L present across mammalian genomes, as revealed by the approximated 60% of homology with the sequence of human ERV-‐L (HERV-‐L) 234. The resulting capsid-‐like protein recognizes specific capsids of MLV strains. The different alleles of Fv1, N and B, differ only in three residues within a small motif associated with their restriction capacity 235 and recognize differentially a residue at position 110 of the amino acid sequence of the CA protein of B-‐ and N-‐MLV, respectively 236. For the binding between the MLV capsid and Fv1 to happen, the gag polyprotein must be mature and cleaved from p12 and NC 237. The direct binding was shown using a biochemical method where capsid-‐coated lipid nanotubes were subjected to immunoprecipitation with Fv1 proteins. As observed by negative staining and electron microscopy, the capsid units assembled in an ordered manner in vitro, dependent on the typical retroviral β-‐sheet formation on the N terminus of the CA protein 238. Importantly, the binding results were in agreement with the specific restriction pattern of the distinct Fv1 alleles 238.
TRIM5
The large family of TRIpartite Motif (TRIM) proteins comprises approximately 100 members in human 239. Besides a few exceptions, they all share the conservation of three main modules in a precise order (reviewed in 240). The Ring-‐finger (RF) domain is found at the N-‐terminal part of a TRIM protein. This
domain is formed of zinc-‐coordinating motifs, that allows the formation of a
“cross-‐brace” structure, involving cysteins and a histidine that contact two Zn atoms 241-‐243.
Present in several families of proteins, RF domains often confer binding to an E2-‐
ubiquitin conjugating enzyme and function as E3-‐ubiquitin ligases to themselves and to other substrates, as reviewed by Deshaies and Joazeiro 243. Indeed, many TRIM family members have been described to display E3-‐ubiquitin ligase activity, including TRIM5, TRIM21 and TRIM25 244-‐246, as reviewed in 240. The nuclear magnetic resonance (NMR) structure of the human TRIM5α RING finger has been solved 247 and showed that the core of the domain is composed of the majority of the hydrophobic residues and is located between two β-‐sheets and an α-‐helix.
As a second motif, TRIM proteins carry one or two B-‐box domains, which share a similar ternary conformation. Indeed, B-‐boxes also coordinate Zn atoms in a crossed configuration and form two β-‐sheets, followed by a α helix, as shown by the study of MID1 (TRIM18) and human TRIM5α 248-‐250. The TRIM B-‐box1 always precedes B-‐box2 and is never found alone 240. In contrast, many TRIMs possess only the B-‐box2, as exemplified by TRIM5 240. The function of B-‐box domains is not completely understood. However, the study of the B-‐box2 of MuRF1 (TRIM62) revealed a surface hydrophobic patch with polar residues on a dimer interface 251. This finding allowed the manipulation of the equivalent residues of TRIM5 B-‐box2 and showed that this domain is important for protein higher-‐
order multimerization 250,252.
As a third motif, the Coiled-‐coil (CC) domain is the last module of the tripartite RING finger-‐B-‐box-‐CC (RBCC) motif. It contains appropriately spaced hydrophobic residues on amphipathic α-‐helices, forming two putative leucine-‐
zipper motives, which are responsible for TRIM protein-‐to-‐protein interactions
253,254. This domain was shown to participate in the higher-‐order multimerization, homo-‐ and hetero-‐dimer formation of TRIM proteins as well as to be required for their concentration into discrete cellular compartments such as the nuclear and cytoplasmic bodies (NB and CB, respectively) 255-‐259.
The TRIM proteins vary in their C-‐terminal domain composition. As reviewed by Ozato and colleagues 240, there are ten distinct C-‐terminal TRIM domains, found alone or in different combinations, and participating to functions as variable as localization to microtubules, binding to a retroviral capsid or interaction with histones and transcriptional repression. The discovery that the TRIM5 gene was the determinant for HIV-‐1 restriction in monkey cells 62,200,201,203, motivated the study of the involvement of TRIM proteins in the innate immunity. Importantly, TRIM25 was found to induce the K63 polyubiquitination RIG-‐I, essential for the RNA viruses-‐triggered signal transduction 245. Although TRIM1, TRIM19 and TRIM22 were already shown to have antiviral properties (as reviewed by Nisole and colleagues 260), a large screen, looking at 55 TRIM proteins revealed that this feature was shared by many other TRIM family members 261. Notably, TRIM11 and TRIM15 were found to restrict the release of HIV-‐1 and MLV, respectively, with TRIM15 recognizing the gag protein in the producer cell in a B-‐box-‐
dependent way 261.
The role of TRIM proteins in the innate immune response is further emphasized by the fact that their expression is up-‐regulated upon type I IFN treatment or induction of TLR by agonists and that they differentially activate the AP-‐1, NFκB and IFNβ promoters 262-‐266.
1.2.4 TRIM5-‐mediated retroviral restriction
Six isoforms have been described for the human TRIM5 gene, namely α, γ, δ, ε, ι and κ 267. Only TRIM5α have been shown to inhibit retroviral replication.
Moreover, TRIM5ι is the second more abundant transcript in some human cell lines and, similar to the isoforms γ, δ and κ, down-‐regulates TRIM5α levels and correspondingly modulates the anti N-‐MLV restriction activity 267. An important particularity of TRIM5α is that it is the only isoform that possess a C-‐terminal PRY-‐SPRY domain (figure 9).
Figure 9: Schematic representation of the TRIM5 orthologues and structure of the proteins. TRIM5 proteins are composed of an RBCC motif, comprising the RING finger (RF), the B-‐Box (BB) and the Coiled-‐
Coil (CC). Some TRIM5 orthologues carry a C-‐terminal capsid-‐binding domain. Whereas most of TRIM5 proteins bear a C-‐terminal PRYSPRY domain, some primate species carry a Cyclophilin A (CypA) module.
The linker 2 (L2) separates the CC from the C terminal domain of TRIM5 proteins.
The PRYSPRY is found on other TRIM5 paralogues and structural studies have revealed that it forms a dimer interface via a donor sequence and an acceptor strand from different protein targets 268. This interface is composed of six variable loops (VLs) that mediate the binding to different specific substrates 269. Importantly, the PRYSPRY domain of TRIM21 recognizes immunoglobulin G (IgG) with contacting residues found in the VL4, where the E405 and E406 of TRIM5α conferring species-‐specific N-‐MLV activity are positioned 269,270.
For its part, the specificity of the restriction of HIV-‐1 by rhesus macaque TRIM5α involves residues in the VL1 271. Indeed, the PRYSPRY domain is the determinant of TRIM5α retroviral restriction specificity 271-‐273 and directly binds to the retroviral capsid 274,275.
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The binding between TRIM5α and particulate capsid protein was never observed. Notably, whereas mature virions could saturate the restriction phenotype, monomeric capsid did not have any effect on retroviral blockade
199,237,276,277. It became later evident that the restriction factor recognized the CA in complexes forming an hexameric lattice 271,274. Soon after viral entry into the host cell cytoplasm, TRIM5α orthologues bind to the retroviral capsid 62,200 and can subsequently mediate a blockade at two steps of the viral life cycle.
The use of single-‐cycle infection assays allows a safe and precise way to determine what steps of the viral life cycle are affected by a cellular factor in non-‐permissive (restrictive) cells. This method uses combinations of three-‐part or two-‐part vectors composed of the viral genome, the packaging genes and an envelope. Once expressed in producer cells, the viral proteins form particles that are used to infect target cells. The challenging of target cells with these virions will result in a single-‐round of infection, given that no complete retroviral genome is provided and thus the virus is replication-‐incompetent. Cells can also be transduced (figure 10), referring to the transfer of DNA by retroviral vectors.
When using bi-‐cistronic vectors coding for a gene of interest and for an antibiotic resistance gene, cells can be selected with the specific antibiotic and give rise to stable cell lines expressing constitutively the gene which function wants to be studied.
First, TRIM5α impedes reverse-‐transcription to proceed, as revealed by the comparison of viral cDNA accumulation between permissive and non-‐permissive cell lines 194,195,202,277. Indeed, the quantification of the reverse-‐transcripts in HeLa cells transduced with a bi-‐cistronic vector revealed that the cells that stably expressed rhesus TRIM5α contained at least ten fold less early and late HIV-‐1 cDNA products than the cells that had been transduced with the vector that only carries the antibiotic resistance gene 62,200.
The TRIM5 gene has been subjected to strong positive selection on residues of the PRYSPRY domain 278,279. In at least two independent events, the cyclophilin A (CypA) cDNA has inserted between exons 7 and 8 or in the 3’ Untranslated
region (3’UTR) of the TRIM5 gene via a LINE-‐1-‐mediated retrotransposition, replacing the PRY-‐SPRY domain and forming a fusion protein (TRIM5Cyp) 62,280-‐
283 (figure 8). Whereas in the case of the New world monkey Aotus trivirgatus (owl monkey), TRIM5Cyp potently blocks HIV-‐1, FIV and SIV from the African green monkey (SIVagm), the version from Macaca mulata restricts HIV-‐2 and FIV
62,282,284.
Similar to TRIM5α, owl monkey TRIM5Cyp binds to the retroviral capsid soon after entry and blocks the reverse-‐transcription 62,285,286. The cylophilin A (CypA) domain is responsible for the binding to HIV-‐1 capsid, as evidenced by the examination of the effect of deletion mutants or the addition of CsA 285,287,288. Although it was shown that the monomeric CA protein p24 could bind to cyclophilins A and B 289, the saturation of the TRIM5Cyp-‐mediated blockade requires completely processed virion cores 68 indicating that the assembled hexameric capsid is the target of the restriction factor.
TRIM5 proteins can additionally target a second and later step of the retroviral life cycle, as evidenced by the treatment of non-‐permissive cells with proteasome inhibitors such as MG132 290-‐293. Indeed, after addition of MG132, an increase in reverse-‐transcripts was observed, but the restriction was still not affected. More profound examination of the abundance of different viral products showed that 2-‐LTR circles, a marker for nuclear import, were affected 291,292, suggesting that a step before integration is also affected by TRIM5 orthologues.
The previous findings allow establishing a two-‐step model of TRIM5-‐mediated retroviral blockade (figure 7). First, inhibition of the reverse-‐transcription is concomitant with the observed proteasome-‐dependent TRIM5-‐mediated disassembly of the capsid 290,293. In the second step, a proteasome-‐independent mechanism is responsible for a block before, or at, retroviral cDNA nuclear import.
1.2.5 TRIM5 is a PRR
The expression of many TRIM proteins has been induced upon type I IFN or TLR agonist treatment 262-‐264,294,295. Importantly, TRIM5α and TRIM5Cyp transcripts are up-‐regulated following the addition of Type I IFN or LPS, in cells expressing the corresponding receptors 262,263,296. Moreover, we found that TRIM5 is required for the establishment of the TLR4-‐mediated antiviral state 265.
In the same study, we showed that TRIM5 function as a PRR for the retroviral core. Notably, TRIM5 synthesizes unanchored K63-‐polyUb chains that activate the TAK1-‐TAB2-‐TAB3 complex leading to the stimulation of MAPK-‐ and NFκB-‐
mediated signaling. Likewise, TRIM5Cyp was additionally able to stimulate AP-‐1 and NFκB promoters. This activity of TRIM5 alone is enhanced when it binds a restriction-‐sensitive capsid. Furthermore, the specific E2 ligase Ubc13 and TAK1 were found to be necessary for TRIM5-‐mediated restriction 265. In agreement with these findings, another study revealed the importance of the E3 ubiquitin ligase function to the TRIM5-‐mediated retroviral blockade, although in contrast with the previous study, autoubiquitination was the proposed limiting process
247. Conversely, a study showed that the E3 ligase function of some TRIM5Cyp is not required for retroviral blockade, as evidenced by the conserved restriction
247. Conversely, a study showed that the E3 ligase function of some TRIM5Cyp is not required for retroviral blockade, as evidenced by the conserved restriction