HIV Pol: does it offer therapeutic targets for HIV?

KEYWORDS:

• HIV
• Pol gene
• CARD8
• CRISPR
• gene editing
• HIV vaccine
• HIV cure

1. Introduction

One of the greatest challenges in the management of HIV infection is the search for new treatment strategies, including those aimed at achieving a functional or sterilizing cure. Although antiretroviral therapy (ART) has dramatically improved the survival and quality of life of people living with HIV (PLWH) over the years, treatment interruption leads to viral rebound; to date, no study has proven to be effective in achieving viral eradication [1,2]. However, increasing knowledge of the HIV genome is enabling the exploration of innovative potential therapeutic approaches.

One of the principal genes of HIV is Pol, that encodes three enzymes required for viral replication: protease (PR), reverse transcriptase (RT), and integrase (IN); all three proteins play a key role in the HIV replication cycle and are therefore targeted by the main drugs in use today [3].

The development of drugs that can inhibit these enzymes has revolutionized the course of HIV infection since the 1990s, and even today the major classes of antiretroviral agents act against protein products of the Pol polyprotein, consisting of nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and integrase strand transfer inhibitors (INSTIs).

ART effectively suppresses the HIV virus, preventing it from replicating and reducing the viral load in the body; the antiretroviral regimens are tailored to each individual based on factors such as viral resistance, potential side effects, and other medical conditions.

Nonetheless, there are still structural elements that are poorly understood, such as the molecular organization of Pol in the Gag-Pol polyprotein: a deeper understanding of the Pol configuration could help in the development of new antiretroviral drugs or the use of strategies such as technologies starting from the Pol gene.

2. HIV Pol as therapeutic target

HIV Pol has been selected as a drug target and studied in past few years in the context of various therapeutic interventions, including the ‘shock and kill’ approach and gene editing. In this editorial, we review relevant papers that present HIV Pol as a key player in the development of antiretroviral strategies, as seen in Figure 1.

Figure 1. HIV Pol as target for different therapeutic strategies.

The innate immune response is crucial to prevent viral invasion or replication in host cell defense against HIV, and understanding of the mechanisms underlying these responses may contribute to design breakthrough treatments.

In recent years, HIV PR has been shown to activate the caspase recruitment domain containing the protein 8 (CARD8) inflammasome, a multiprotein complex responsible for caspase-1 (CASP1)-mediated cell apoptosis. CARD8 is normally expressed in HIV target cells, including CD4 lymphocytes and macrophages, and its cleavage by HIV PR is critical for CARD8 to interact with CASP-1 and trigger pyroptosis in infected cells [4,5].

However, rapid cell death can be induced by premature activation of PR that results from overexpression of PR or use of some NNRTIs, such as efavirenz and rilpivirine, due to binding to HIV Pol and enhancing Gag-Pol dimerization. PR specifically cleaves the N-terminus of CARD8 and the neo-N-terminus is then degraded by proteosomes [6]. This process results in the release of the bioactive UPA-CARD fragment and therefore to the activation of the CARD8 inflammasome, leading to pyroptosis in HIV-infected cells [6]. In the absence of these conditions, PR catalytic activity is low and HIV-infected cells are able to evade CARD8-induced death [7].

Animal models have been investigated to better study the influence of CARD8 in HIV infection. In humanized mice reconstituted with CARD8-deficient cells, CD4 reduction was delayed despite high values of HIV RNA; confirming these data, it has been showed that in SIV-infected rhesus macaques and sooty mangabeys, CARD8 can be responsible for CD4 T cell depletion [8].

Novelties have also been reported in literature regarding the molecular organization of Pol.

Harrison et al. produced a stable version of HIV Pol and its three-dimensional structure was investigated by cryogenic electron microscopy (cryo-EM) [9]. Thanks to the results of this study, it has been possible to better define both the function and the architecture of the Pol precursor prior to the PR cleavage: indeed, the formation of RT dimers seems to be a first step in PR activation by positioning the monomeric PRs into close proximity, that subsequently promotes PR dimerization.

At the structural level, PR and RT are strictly linked: the proximity of the N termini of the two RTs causes the two PR monomers to be attracted to each other. It is not yet known how the IN domain interacts, as the model under study did not describe how IN is arranged within Pol, probably as a consequence of the flexibility between RT and PR [9]. While cryo-EM excels at determining the static structures of biological macromolecules, innovative techniques are still needed to unravel the dynamics of certain proteins.

Therefore, these original insights into the structure of the Pol polyprotein, based on the evidence that the maturation of HIV Pol precursors is largely influenced by the dimerization of RT, may help in the development of new antiretroviral drugs. Because polyproteins can be long-lived and have distinct sequences at their processing junctions, they can form unique inhibitor binding sites that are distinct from mature proteins. By targeting these sites, researchers may be able to develop drugs that interfere with early events in HIV maturation, potentially disrupting the viral life cycle.

Moreover, HIV Pol is one of the most highly conserved regions in the genome of the virus and for this reason it has been identified as target for clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing approach [10]. However, HIV was able to escape viral inhibition: indeed, non-homologous end joining is the main pathway for repairing DNA double-strand breaks and it can result in nucleotide substitutions, insertions, and deletions at the cleavage site [11].

To overcome this issue, guide RNAs targeting long terminal repeat regions were combined with guide RNAs targeting Gag and Pol [12,13]; this approach yielded more promising results, in particular when Nguyen et al. designed a guide RNAs that targeted four different highly conserved regions, including the active site of PR, the catalytic core of IN, and the c-terminal domain of IN in the Pol gene: In this way, it has been shown that more than 90% of the inhibition of HIV can be achieved in vitro [14].

Further studies of CRISPR technology with different Cas systems are needed to achieve long-term suppression of HIV replication in vivo in the context of a cure for HIV, likely through a multiplex method able to target the virus with two or more guide RNAs versus one or more genes.

Lastly, Pol has previously been investigated in the field of HIV vaccines [15,16]. Recent models have shown that a reduction in reverse transcription capacity is linked with cytotoxic T lymphocyte escape-associated RT and IN mutations that compromise viral fitness; therefore, these variants in Pol may be targeted for the design of an attenuation-based HIV vaccine [17]. A previous work described the use of a self-amplifying mRNA vector in a murine model to administer a mosaic comprising six highly conserved regions of HIV gag and pol [18]. Study findings revealed that this vaccine could trigger an enduring CD4 and CD8 T-cell response in mice that received the self-amplifying mRNA vector initially and were later bolstered with a modified vaccinia virus Ankara viral vector [18]. A robust T-cell response, the elicitation of neutralizing and functional non-neutralizing antibodies, and a reduction in acquisition of infection were induced in animals vaccinated with Adenovirus (Ad)/modified vaccinia Ankara or Ad/Ad vector-based vaccines expressing bivalent HIV mosaic env, gag, and pol [19].

3. Expert opinion

Although the study of the HIV genome began many years ago, modern technologies are still enabling us to discover important new details in the structure of proteins involved in viral replication and provide more knowledge about the processes behind virion maturation.

New insights into the architectural organization of HIV pol open up the possibility of designing new drugs. Even though current antiretroviral regimens are effective in the maintenance of virologic suppression and have a low toxicity profile, the possibility of using new antiretroviral agents in clinical practice could be a great opportunity for those PLWH with limited treatment options, such as people harboring a multidrug-resistant virus, to ensure that therapy is tailored to individual needs.

Furthermore, Pol could also play a key role in the development of various strategies and be the focus of innovative experimental studies that aim to achieve the goal of HIV remission or eradication. Indeed, treatment strategies still under investigation, such as gene editing, may use genes as Pol on the path to an HIV cure; given that Pol gene is a highly conserved region of the HIV genome and its protein products are essential in the progression of HIV infection, it is a valid target for CRISPR-Cas system.

In conclusion, the opportunities for successful antiretroviral approaches to continue to target Pol are considerable thanks to the scientific progress made due to the latest advances in technology.

Article highlights

• Advances in knowledge of the structure of HIV Pol may contribute to the development of antiretroviral strategies.

• The dimerization of reverse transcriptase has a profound effect on the maturation of HIV pol precursors.

• An approach targeting CARD8 inflammasome activation combined with a ‘shock and kill’ scheme may be explored for an HIV cure trial.

• Because Pol is a highly conserved region of the HIV genome, it can be targeted by CRISPR-Cas technology and designed for HIV vaccine.

Abbreviations

PR, protease; RT, reverse transcriptase; IN, integrase.

Declaration of interests

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.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.