Innate Barriers to Genome Invasion

Over the course of evolution, retroviruses and their hosts have been in constant conflict, resulting in a long-term evolutionary "arms race" in which host and pathogen alternately gain selective advantages through functional innovations. On the host side, cells of higher eukaryotes have evolved to encode numerous inhibitory activities called restriction factors to block retroviral replication. Conversely, retroviruses have evolved to exploit numerous host cellular components to replicate and to efficiently counter host intrinsic lines of defence. The current acquired immunodeficiency syndrome (AIDS) pandemic exemplifies this struggle, as its causative agent the human immunodeficiency virus (HIV) was found to overcome several host-encoded restriction factors active against other retroviruses. TRIM5α is one such restriction factor, which inhibits retroviral infection soon after the virus enters its target cell by recognizing the capsid of incoming virions through its C-terminal PRYSPRY domain. Different TRIM5α alleles block distinct retroviruses in a species-specific manner: Human TRIM5α (huTRIM5α) potently blocks the so-called N-tropic murine leukemia virus (N-MLV), but is ineffective against the closely related B-tropic (B-MLV) and Moloney (Mo-MLV) strains, or against the simian immunodeficiency virus from macaque (SIVmac) and the two HIV strains (HIV1 and HIV2). In contrast, rhesus macaque TRIM5α (rhTRIM5α) potently blocks HIV1 and HIV2 but not N-, B-, Mo-MLV or SIVmac. My thesis aimed at gaining insight into the molecular mechanism of TRIM5α-mediated retroviral restriction. Firstly by investigating the huTRIM5α specific capture of retroviral capsids to better understand the ability of HIV1 to escape this recognition, and secondly by identifying cellular factors that interact with TRIM5α to subsequently determine their putative role in TRIM5α-mediated antiretroviral activity. In the first set of experiments, I demonstrated that substituting a single amino acid in the PRYSPRY domain of huTRIM5α expands its antiviral activity to B-MLV, Mo-MLV, SIVmac and HIV2, while HIV1 is still able to escape this block. Furthermore, I found that MLV could escape this restriction through the negative influence of four critical residues within its capsid, supporting a model whereby TRIM5α directly interacts with retroviral capsids. In the second part of my thesis, I exploited the remarkable conservation of the capsid among retroviruses to map the four key positions found in MLV onto the HIV1 capsid. I then confirmed that amino-acid substitutions at some of these positions could increase the sensitivity of HIV1 to huTRIM5α restriction. Further mutating an exposed cyclophilin A-binding loop of the HIV1 capsid could reinforce susceptibility to this factor. These results indicate that, despite major sequence divergences, HIV and MLV use structurally homologous residues to avoid capture by huTRIM5α. I then investigated the influence of the coiled-coil domain of huTRIM5α on the range of retroviruses recognised by this antiviral factor. I found that the PRYSPRY domain is necessary but not sufficient to dictate TRIM5α specificity as single amino acid changes within the coiled-coil region could modulate the range of targeted retroviruses. We favour a model whereby the coiled-coil domain exerts this effect indirectly, by influencing the orientation of the PRYSPRY domain and hence its ability to recognize retroviral capsids. Finally, I screened a human cDNA library for cellular proteins that interact with huTRIM5α. I found several hits, the physiological roles of which impact on cellular processes as diverse as ubiquitination and endocytosis. These results open possible avenues to pursue in future studies aimed at deciphering further the mechanisms by which TRIM5α blocks retroviruses.

The Interplay between Cytidine Deaminases, HIV and Domesticated Genetic Parasites

"There is more virus in us than us in us". John Coffin's famous sentence illustrates that particular nucleic acid sequences related to exogenous viruses, called retrotransposons, constitute almost half of the human genome and largely exceeds the amount of protein-coding DNA (section 1.4). At the beginning of the past century, biologists realized the size of the genomes of various eukaryotes did not correlate with the level of complexity of these species. In extreme cases, certain species such as the fish L.paradoxa had 30-times more genomic DNA than do primates. In 1980, Francis Crick and Carmen Sapienza independently postulated that part of this once-called "junk DNA" must stem from self-replicating units, categorized as "selfish DNA" to distinguish them from more "altruistic" cellular genes. Indeed, while genes amplify in a population because of the selective advantage they confer to their host, the selfish DNA amplifies without making any contribution to the host phenotype and survives rather as a not-too-deleterious parasite. In early 2000's, the accomplishment of the human genome sequencing projects gave formal credence to Crick's and Sapienza's initial hypothesis. Retrotransposons replicate in the genome of their host by a copy-and-paste mechanism. Accordingly, any new retrotransposition event causes a genetic alteration potentially detrimental to the host. On an evolutionary perspective however, accumulation of retrotransposon-derived sequences can be beneficial to the species after these once "selfish" elements become "domesticated" to deserve more physiological roles. In human or mouse for instance, retrotransposons are known to cover a large spectrum of cellular functions, from gene regulation to antiviral defense (section 1.6). In order to maintain under tight control the balance between the good and adverse effects of retroelements, eukaryotes have evolved cellular defenses aimed at inhibiting the replication of such elements (section 1.7). Gene silencing by DNA methylation is a mechanism widely used in plants, fungi or vertebrates to control the expression of self-replicating elements. For instance in mammals, specific KRAB-zinc finger proteins can target retroviral-derived sequences and mediate their transcriptional silencing (section 3). The first aim of this study was to characterize the sensitivity of retroviruses to KRAB-mediated epigenetic silencing in varied chromosomal contexts (section 5). Since some retroviruses like HIV favor integration within transcriptionally active regions, these viruses may escape blockade by integrating chromosomal area refractory to epigenetic silencing. We found that instead, KRAB-mediated repression mechanism was not dependent upon integration site of a retroviral vector, hence the emergence of viruses escaping KRAB-mediated repression would be unlikely. Mammalian cells are also able to control the replication of retrotransposons at a post-transcriptional level via the APOBEC3 family of cytidine deaminases (section 2). Since certain retrotransposons are related to exogenous retroviruses, it is perhaps not surprising that APOBEC3 proteins can also inhibit a range of such elements including HIV. While there is only one APOBEC3 gene in the mouse genome, the family considerably expanded in other species such as primates were it comprises seven members (section 2.1). Although it is tempting to postulate that the expansion of the family in primates was driven by the strong selective pressure imposed by various genetic threats growing in number and complexity, it is not understood how only a few homologous proteins can target such a disparate group of replicating elements. The second aim of this study was to understand the mechanism by which different APOBEC3 proteins can target and inactivate particular self-perpetuating elements (section 4). We launched an integrative study focused on two human APOBEC3 homologues with distinct antiviral functions. APOBEC3A, a single-domain cytidine deaminase, can block efficiently the parvovirus adeno-associated virus type 2 (AAV-2) or the retrotransposon LINE-1 but cannot inhibit HIV replication. APOBEC3G is formed of two independent cytidine deaminase domains, only the C-terminal of which is catalytically active. APOBEC3G is endowed with restriction activity against HIV, but is largely ineffective against AAV-2 or LINE-1. We combined functional studies with structural modeling and phylogenetic analyses and found that restriction in each case involved similar regions at the surface of the protein and nearby the catalytic center, yet with distinct shapes and nucleic-acid interacting properties between the homologous proteins. While on APOBEC3G, the amino-acids in this region mediate intersubunit contacts between monomers and the predicted dimer interface shapes a groove that may facilitate binding to single-stranded RNA, on APOBEC3A however, it is corresponding residues on the monomer that form a DNA-binding groove important for editing. In summary, this study describes the nucleic-acid-interacting domains of APOBEC3A and APOBEC3G essential for their intrinsic restriction activities, and further suggests that structural variations in this domain may contribute to define the sets of targets inhibited by each APOBEC3 protein. APOBEC3 proteins may be considered as modular units, whose restriction function can evolve with emerging selective pressures by specific variations in their nucleic-acid binding domain, alternatively by fusion of two APOBEC3 proteins endowed with different functionalities. APOBEC3 proteins would efficiently protect the genome integrity of their host against highly variable genetic threats or "selfish DNA", in combination with other restriction factors such as the KRAB-zinc finger proteins. Contrary to epigenetic silencing however, post-transcriptional restriction as provided by APOBEC3 proteins may prohibit the deleterious effect of retrotransposition, without altering other retroelement functions putatively important for the physiology of the cell.

EPFL2010

Homology-based identification of capsid determinants that protect HIV1 from human TRIM5α restriction

Pierre Maillard, Olivier Michielin, Didier Trono

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.

American Society for Biochemistry and Molecular Biology2010

Polynucleotide cytidine deaminases, innate immunity and hepatitis B infection

Stéphanie Jost

More than 350 millions individuals worldwide suffer from chronic hepatitis B virus (HBV) infection, a condition that evolves towards liver insufficiency and hepatocellular carcinoma in approximately 15 to 40 percent of cases. Yet the majority of de novo HBV infections, except for those occurring during the perinatal period, are cleared during the acute phase. This largely appears to result from non-cytopathic host defense mechanisms involving cytokines and notably interferons (IFNs). A key role for IFNs in the biology of HBV infection has been further illustrated by studies in animal models of the disease. Besides, IFN-α is the standard treatment for chronic hepatitis B. Although type I and II IFNs are the most important immune-regulators of HBV replication, the downstream effectors mediating the inhibitory action of IFNs on HBV replication have not yet been identified. In a first place we showed that HBV replication is blocked by human APOBEC3G and APOBEC3F, two closely related editing enzymes belonging to the family of polynucleotide cytidine deaminases, whose catalytic activity results in the conversion of cytidine residues to uridine residues. Both proteins were initially recognized for their ability to inhibit retroviruses, a property which mainly depends on their editing activity, and as the targets of the virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV), which induces their degradation. The family of polynucleotide cytidine deaminases also comprises Activation-Induced cytidine Deaminase (AID), APOBEC1, APOBEC2, and the products of the APOBEC3A through APOBEC3H gene cluster. AID is essential for class switch recombination and somatic hypermutation of the immunoglobulin locus in pre-B lymphocytes, thus for the generation of antibody diversity, and APOBEC1 edits the apolipoprotein B mRNA in the guts, thereby regulating cholesterol metabolism. By comparison, little is known about the physiological functions of the other family members. However, apart from APOBEC2 and APOBEC3A, sequence analysis of the genes encoding these enzymes suggested that they possibly evolved as a host defense against pathogens. This hypothesis was confirmed for most of APOBEC3 proteins, which were independently found to restrict the replication of various exogenous and endogenous retroelements, as well as the adeno-associated virus. For this reason, we next explored the range of cytidine deaminases active on HBV and found that besides APOBEC3G and APOBEC3F, APOBEC3B, AID and APOBEC1 are also potent inhibitors of HBV. In parallel, while examining in details the mechanisms underlying the antiviral activity of APOBEC3G against HBV, we found that this cytidine deaminase blocks HBV infection not by lethal editing of nascent reverse transcripts, as for retroviruses, but rather by impairing the accumulation of pregenomic RNA (pgRNA)-containing viral capsids, the subviral structures in which HBV reverse transcription takes place before virions containing a partially double-stranded DNA genome are released. Remarkably, this is similar to the decreased steady-state levels of pgRNA-containing capsids which are observed upon exposure of HBV infected cells to IFNs. It suggested that IFNs and cytidine deaminases might affect HBV replication through the same pathway. The first step to unveil the in vivo relevance of antiviral cytidine deaminases for the control of HBV replication consisted in our demonstration that type I and II IFN treatment of human hepatocytes induces the production of APOBEC3G, and to a smaller extent, that of APOBEC3F and APOBEC3B, but not that of AID and APOBEC1. These observations led us to ask whether the induction of antiviral cytidine deaminases by cytokines contributes to the IFN-induced clearance of HBV, as IFN-α and IFN-γ are both found in high amounts in the liver of acutely infected individuals. Importantly, we revealed that blocking APOBEC3G, APOBEC3F and APOBEC3B in hepatic cell lines does not abrogate the inhibitory effect of IFNs on HBV, indicating that antiviral cytidine deaminases are not the main mediators of the action of IFNs on this pathogen. Nevertheless, while HBV replication was not impaired in hepatic cells when these enzymes were expressed at their baseline levels, they could edit the HBV genome in these conditions, suggesting that cytidine deaminases might participate in the emergence of new HBV strains, notably those that have been recognized as being of clinical importance. Finally, the results of this work indicate that a therapeutic strategy based on the expression of antiviral cytidine deaminases in the liver could be envisioned to cure HBV chronic infections.

EPFL2007

The Interplay between Cytidine Deaminases, HIV and Domesticated Genetic Parasites

EPFL2010

Polynucleotide cytidine deaminases, innate immunity and hepatitis B infection

Stéphanie Jost

EPFL2007