Harnessing the therapeutic potential of host antiviral restriction factors that target HIV

Keywords::

• antiviral therapy
• APOBEC3G
• HIV
• restriction factors
• tetherin

The development of highly active anti-retroviral therapy (HAART) against HIV and the associated disease in HIV-driven morbidity and mortality is arguably one of the most successful medical interventions of recent years. Yet despite this success, the HIV pandemic remains a significant global disease burden. Despite promising new approaches, there is still no prospect of either an effective vaccination strategy or a practical curative strategy in the near future. Further, HAART is compounded by the inevitable development of viral resistance to drugs, a consequence of the high reproductive and mutagenic rate of HIV in the context of strong selective pressure. For these reasons, the development of new classes of antivirals is necessary to maintain therapeutic options for patients who have developed resistant viruses. Equally, the prospect of antiviral-based pre-exposure prophylaxis regimens, or topically applied prophylactic microbicides, will require a broadening of the antiviral repertoire to minimize rates of transmitted resistance.

Currently, the majority of the viral life cycle and enzymatic processes are targeted by HAART, including virus entry, reverse transcription, integration and maturation. With such comprehensive coverage, comes the necessity to identify new targets.

Engendered by the wide-spread implementation of genomics techniques, one of the central themes of basic retrovirology research in the last decade has been the realization of the profound scale of interactions between the HIV virus and the host cell. For example, a number of genome-wide studies have highlighted the extent to which HIV uses factors present in host cells to complete its replication cycle [1]. Yet even prior to this, it was known that cells express dominant factors that can antagonize viral replication, termed restriction factors. Restriction factors are typically interferon-induced proteins that when expressed as single gene products can antagonize viral replication, often reducing it by orders of magnitude. Therefore, viruses typically express countermeasures to them (for a review, see [2]). There is now ample evidence that restriction factors are active in animal models and patients [3]. While modifying cellular factor function directly may lead to cytotoxic off-target effects, inhibiting viral countermeasures to restriction factors might not, and should render virus susceptible to their antagonizing effects. Due to these attractive properties, there is much interest in developing new therapeutic strategies based on the antiviral properties of restriction factors.

APOBEC3G

APOBEC3G was defined based on the permissiveness of certain cell lines to HIV-1 bearing the accessory protein vif. In APOBEC3G expressing cells, APOBEC3G can be packaged into virions along with viral genomic RNA leading to virus mutagenesis via APOBEC3G-mediated cytosine to uracil deamination during reverse transcription. This results in mutated defective viral genomes and subsequently reduced viral replication [2]. Other APOBEC3 proteins such as APOBEC3F have also been found to have antiviral effects, while more direct effects of APOBEC3 proteins on virus replication have also been noted. HIV overcomes APOBEC3G by expressing the accessory protein vif, which targets APOBEC3G by polyubiquitination leading to its proteasomal degradation, and so produces reductions in the steady state levels of APOBEC3G. However, other vif-mediated mechanisms may also exist.

Therapeutic interventions targeting vif could exploit the mutagenic capacity of APOBEC3G leading to hypermutation of virus resulting in lethal viral genome instability [4]. Development of inhibitors was initially difficult owing to a lack of precise data on vif structure, yet functional approaches have been very fruitful in characterizing vif domains, especially when combined with the knowledge of the APOBEC3G crystal structure. Impressively, only 6 years stood between the first description of the antiviral function of APOBEC3G (2002) and the identification of a small molecule vif antagonist (2008). A compound screen identified the drug RN-18, which leads to vif degradation in the presence of APOBEC3G, yielding decreased viral replication in APOBEC3G expressing cells [5]. Another compound screen identified two molecules that appear to function by reducing vif-APOBEC3G protein–protein interactions, resulting in reduced polyubiquitination of APOBEC3G by vif [6].

An alternative means of exploiting the vif–APOBEC3G relationship is to induce virus hypomutation rendering the virus incapable of mutating in the face of selective pressure [4]. This hypothesis arises from observations that a proportion of HIV variability can arise from APOBEC3G-mediated mutagenesis [7,8]. If the virus is hypomutated, it may become unable to evolve away from immunological or antiviral selective pressures, thus a hypomutating drug may be a useful therapeutic adjunct. Therefore, one study screened a compound library and identified a series of direct inhibitors of APOBEC3G function [9]. The authors were able to derive significant mechanistic insight using the APOBEC3G crystal structure, leading to suggestions that these compounds function via competitive inhibition at the APOBEC3G active site. However, the function of these compounds in viral assays remains to be defined; new in vivo experiments will be required to understand the feasibility of hypomutation as a means of therapy.

Given these initial successes, a debate has emerged as to the likelihood of vif inhibition being successfully included within HAART. Uniquely, as APOBEC3G can alter the mutagenic rate of HIV, there are concerns that hypermutating drugs might not attain inhibitory levels of viral genome instability and so might lead to increased rates of drug resistance. Indeed, there are reports that APOBEC3 might aid the emergence of some reverse transcriptase inhibitor mutations [10,11]. Yet bioinformatic analysis suggests that relatively few common sites of drug resistance mutations are susceptible to APOBEC3-mediated mutation, although such analysis cannot control for the possibility that the virus will pursue alternative undescribed resistance pathways during vif inhibition [12]. Therefore, the future of vif-APOEBEC3 drug development will necessarily depend on careful analysis of the emergence of antiviral drug resistance in the context of HAART.

Tetherin

Much as our understanding of APOBEC3G arose from understanding of defects associated with virus lacking an accessory protein gene, so too did our understanding of the antiviral restriction factor tetherin. It was noted in certain cell lines that HIV particle release was antagonized when HIV did not express its accessory protein vpu. Subsequent analysis identified tetherin (BST-2/CD317/HM1.24) as the host protein that underlies the phenotype. Tetherin is a type II class single pass membrane protein whose antiviral function seems to arise from its structure, allowing virus to be tethered to it on the cell surface [2]. HIV-1 evades tetherin by expressing the accessory protein vpu, with the weight of evidence suggesting that vpu reduces cell surface expression of tetherin. While the exact mechanism(s) involved is still debated, ultimately vpu expression rescues viral release. As vpu exerts its effect on tetherin via direct interactions, it too provides opportunity for therapeutic intervention.

Currently, efforts are less developed than for APOBEC3G, reflecting the more recent discovery of tetherin. There were early proposals suggesting disruption of vpu–tetherin interactions on the cell membrane by using tetherin peptide mimics as competitive vpu inhibitors [13]. Though the current state of development for this method is unknown, it should be possible to develop small molecule inhibitors based on improved structural knowledge. Assays to define vpu-tetherin protein–protein interactions and their antagonism are relatively tractable and will be useful in high throughput screening approaches [14].

Although hope emerged from the finding that an antagonist (BIT225) of the ion-channel activity of HIV-1 vpu (a process thought to be distinct from its anti-tetherin activity) was shown to antagonize viral release in human macrophages [15], suggesting an effect of the drug on tetherin-mediated viral antagonism, further analysis in CD4⁺ T-cell lines found that there was no effect on either cell surface tetherin expression or on HIV particle release [16]. These data suggest that the antiviral activity of BIT225 might exclusively arise in macrophages and/or via vpu ion-channel inhibition.

TRIM5α

Understanding of the human TRIM5α restriction factor arose from understanding of the pattern of retrovirus restriction phenotypes found in mice that target specific retroviral capsids [2]. Ultimately, this led to a cDNA library screen that identified TRIM5α as the gene responsible for a postentry capsid-mediated restriction of HIV-1. TRIM5α is a cytoplasmic protein that interacts with incoming viral capsids leading to inhibited viral reverse transcription. Yet a complete understanding of TRIM5α-capsid interactions or the overall mechanism of restriction is still lacking and is the source of intense debate. This has led to the proposal that multiple mechanisms might govern TRIM5α function, although it is yet not clear if this makes therapeutic intervention based on TRIM5α a more difficult proposition or if a single intervention might be able to target multiple pathways.

Yet, substantial progress has been made in work with the related restriction factor TRIMCyp, a TRIM5α derived protein containing a cyclophillin-A motif, found naturally in rhesus macaques and other simians [17]. TRIMCyp possesses potent activity against HIV-1, whereas HIV-1 capsid is naturally resistant to human TRIM5α. Detailed structural understanding has allowed the development of re-engineered TRIMCyp proteins with a conformation specific for improved activity against HIV [18,19]. Using gene therapy-based approaches such proteins have already been shown to be effective against HIV in humanized mouse models [19]. Furthermore, the detailed structural data arising from such analyses will also be of high value in devising small molecule based approaches. Notably, one compound (PF74) that was found to destabilize the HIV capsid demonstrates significant antiviral effect [20]. It is thought that this function may mimic that of TRIM5α. This avenue of research is exciting, as it offers a more readily available and affordable, though admittedly transient, means of targeting the HIV capsid, while gene therapy-based approaches are being optimized

Conclusions

Currently, there are no restriction factor-based antivirals in clinical development, but the rapid progress from their identification to functional understanding and preliminary evaluation of antiviral strategies is impressive. From the three examples of APOBEC3G, tetherin and TRIM5α, it is clear that a solid foundation of functional and structural data is required for progress toward therapeutics, as exemplified by the host–cell virus chromatin-targeting factor LEDGF/p75 that has allowed the rapid and rational design of small molecule antagonists of this virus–host interaction [21]. Although some progress is perhaps seen in the case of APOBEC3G, advances in regard to tetherin and TRIM5α are also apparent. While gene therapy approaches based on restriction factors may also be viable [22], it is unlikely that such measures will be possible in resource-limited settings, where the burden of HIV disease is the highest.

While the rationale for targeting virus countermeasures to restriction factors is sound (limiting off-target effects), it is still not clear if disrupting the delicate host–pathogen balance of broad-range restriction factors might be problematic in vivo. For example, APOPBEC3G also affects endogenous retroelements that undergo reverse transcription; it is unknown what the cellular and immunological consequence of altering their expression levels via therapy may be.

Yet, there are ever more prospects as new antiviral restriction factors are being identified with increasing frequency. SAMHD1 was recently found to be a restriction factor in myeloid cells that controls HIV replication and that can be overcome by the HIV-2 vpx gene. Screens for new restriction factors have identified the interferon induced transmembrane proteins as antagonists of a wide range of unrelated viruses, including HIV-1 [23,24]. In the long term, it may be possible to derive both virus-specific drugs as well as broad-spectrum antiviral agents based on an understanding of broad-spectrum antiviral restriction factors [25]. The list of restriction factors is likely to grow, so practical progress will be achieved even if only a minority of them yield antivirals.

Acknowledgements

The authors thank Dr Chen Liang and Aaron Donahue of the McGill AIDS Centre for their helpful comments on this manuscript.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.