HIV – Repliclones can grow large enough to neutralizing antiretroviral therapy

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Increasingly, UPMC’s chief of infectious diseases – a well-regarded expert in HIV/AIDS – is contacted by a perplexed physician describing a patient with HIV who insists they are adhering to the daily medication regimen meant to keep the virus in check, but testing says otherwise.

Virus is still showing up in the patient’s blood, something clinicians believe can’t happen when the infection is controlled with medication. University of Pittsburgh School of Medicine scientists report today that they’ve solved the mystery – and the answer has clinical implications.

In a study published in the Journal of Clinical Investigation, Pitt infectious disease researchers show that the issue isn’t nonadherence to medication or resistance to the drugs.

Instead, the patients are victims of what the scientists have dubbed “repliclones” – large clones of HIV-infected cells that produce infectious virus particles.

“We found that repliclones can grow large enough and produce enough virus to make it appear that antiretroviral therapy isn’t working completely even when it is,” said senior author John Mellors, M.D., who holds the Endowed Chair for Global Elimination of HIV and AIDS, and is chief of the Division of Infectious Diseases at Pitt and UPMC.

HIV replicates by taking over a cell’s machinery and using it to produce more virus, which can then go on to infect other cells. Antiretroviral therapy, which is taken daily, prevents the virus from infecting new cells so that even though HIV can’t yet be cured, it can be controlled to the point that it isn’t detectable in blood tests.

Elias Halvas, Ph.D., research assistant professor in Pitt’s Division of Infectious Diseases, and Mellors led a multidisciplinary team of U.S.-based HIV scientists in investigating the medical records and blood from eight patients with non-suppressible HIV viremia – detectable virus in the blood – despite adherence to antiretroviral medications. Repeated samples of each patient’s blood revealed identical viral genetic sequences that did not change over time.

“This indicates that, in the individual patients, the virus in their blood was coming from identical cellular factories,” said Halvas.

In short, rather than the virus going out and infecting new cells, already infected HIV-producing cells are growing into large clones that make and release virus.

Current medications for HIV infection block the virus from infecting new cells but don’t affect virus production from cells or clones of cells that are already infected.

“Even though we don’t have evidence that the virus produced by these repliclones is then infecting new cells – which would be detrimental to the patient’s immune system— – they could cause other problems, such as chronic inflammation,” said Mellors, who also is Distinguished Professor of Medicine at Pitt.

“If the patient were to stop drug therapy, the virus could have a head-start on rebounding. And repliclones are a key barrier to developing a true cure for HIV.”

The immediate implication of their discovery, Mellors said, involves informing clinicians and patients that HIV viremia can be caused by repliclones.

This can help clinicians in developing disease management plans that may allow continuation of current antiretroviral regimen, knowing that switching the treatment may not suppress the viremia. Instead, the patient can be monitored over time for changes in the level of viremia, which can decline as the repliclone shrinks or sometimes can stay the same or increase slowly. Large increases in viremia should prompt the review of medication adherence again and exclusion of new drug resistance, Mellors added.

For the long-term, scientists must figure out how repliclones escape the immune systems and how they can be efficiently killed to cure the infection. While more research is needed, Mellors and his team speculate that smaller, less easily detected repliclones may be present throughout the body and be responsible for the rapid rebound of HIV in patients who stop their therapy. An added complexity is that all the cells of a repliclone may not all be making virus at the same time and thus remain hidden from the immune system as a latent or invisible reservoir of HIV.

“Many scientists around the globe are working hard to expose the HIV reservoir and destroy it,” said Halvas.


HIV-1 is a complex retrovirus belonging to the Lentivirus genus and its genome is approximately 9.5 kb long. It is composed of nine genes, which encode 3 different types of proteins: structural proteins (Gag, Pol and Env), regulatory proteins (Tat and Rev) and accessory proteins (Nef, Vif, Vpr and Vpu). These proteins are translated from a single sense unspliced or alternatively spliced transcript, which is initiated from the typical retroviral promoter located at the 5′ LTR.

Antisense transcription is not an uncommon phenomenon in retroviruses, since antisense transcripts coding for proteins have been characterized for each member of the Human T-cell Leukemia Virus (HTLV) family (for further information, see [1]).

As early as 1988, a study suggested the existence of an HIV-1 open reading frame (ORF) found on the antisense strand [2]. Subsequent to this study, a few studies have addressed the detection and characterization of this Antisense Protein (ASP), but its existence has remained controversial for many years.

Our recent studies however demonstrated the presence of ASP and further provided a potential role in autophagy and in HIV-1 replication. This review focuses on recent findings on ASP and on future perspectives associated with these findings.

First evidence for the existence of this new HIV-1 protein

The occurrence of HIV-1 antisense transcription was suggested in 1988, a few years after the discovery and the isolation of the virus. This first study consisted of an in silico analysis of 13 HIV-1 strains and provided solid arguments in favor of the existence of a new HIV-1 protein produced from the complementary strand of the env gene [2].

Firstly, this ORF, termed ASO1 (AntiSense ORF1) harboured a high degree of conservation in amino acid sequence.

In fact, 12 of the 13 strains contained this antisense ORF, while the outlying strain was known to be non- infectious.

Interestingly, the amino end was the most conserved region showing complete homology in the first 83 amino acids. Secondly, the ASO1 ORF was not in frame with that of Env, suggesting that the presence of this antisense ORF could not be attributed to selective pressure for the maintenance of a functional env gene.

Furthermore, the ASO1 ORF had been noted to span a sequence of more than 500 bp, which essentially excluded a potential non-selective process driving the conservation of this ORF. Finally, a potential antisense promoter region and polyadenylation signal found downstream of the ASO1 ORF was suggested [2].

In light of this study, researchers first focused on the detection of the antisense transcript and the characterization of the antisense promoter. However, the main difficulty in studying this antisense gene was its poor expression in HIV-1-infected cells, and in fact, this issue became an important obstacle for subsequent studies.

This convincing data over potential functions for this protein [3]. This is thus equally applicable to the HIV-1 asp gene.

HIV-1 antisense transcription and its associated promoter

The first detection of the ASP transcript came from a study by Burkinsky et al. in which, by Northern blot analyses, the authors revealed the presence of three poorly abundant transcripts of 1, 1.1 and 1.6 kb long [4].

Michael et al subsequently isolated cDNAs from chronically HIV-1-infected A3.01 cells, and by using standard RT-PCR analyses, further supported the detection of HIV-1 antisense transcripts [5]. However, a major control was lacking. Indeed, endogenous RT priming, which results from non-specific RT priming by RNA fragments or degraded viral DNA might have been likely responsible for most of the amplified signals.

Thus, it was not possible to determine the strand specificity of the detected amplicon [5]. In this study, transcription initiation sites were identified at the 3′ LTR in addition to a polyadenylation site, which was potentially located at the 3′ end of the ASP ORF.

Finally, analyses of the presumed antisense promoter in the 3′ LTR highlighted an important role played by NF-B, USF and Sp1 [5, 6]. Furthermore, in this study, the Tat protein was presented as a negative regulator of the antisense promoter [5].

Two subsequent studies shed light on the ability of the 3’ LTR to act as an antisense promoter, although it was strongly suggested that its activity was much less active than the 5′ LTR sense promoter [7]. The presence of an initiator element (Inr) in the antisense promoter has also been suggested to contribute to the antisense transcription initiation [8].

This latter study allowed the detection of antisense transcripts by the use of biotinylated primers located at the 3′ LTR.
In a subsequent study, we re-addressed the characterization of the HIV-1 antisense transcript overlapping the ASP ORF in different infected cell lines [9].

Although previous studies suggested the existence of such antisense transcripts, using appropriate controls, we were also successful in specifically detecting this transcript by using standard antisense transcript-specific RT-PCR, in which the added primer in the RT step had a 3’end specific to the antisense transcript and a 5’ non-complementary end targeted by the reverse primer during PCR amplification.

This approach ensured that amplified products could only be derived from cDNA synthesized by the RT primer. Under our experimental conditions, the length of the antisense transcript was estimated to be 4.1 kb. Furthermore, our antisense transcript-specific RT-PCR approach suggested that these transcripts were more abundant in monocytic than in T cell lines [9], although these differences were not quantitatively measured.

By RACE analyses, multiple transcription initiation sites were identified upstream of the ORF of ASP and no spliced forms were detected in these antisense transcripts (Figure 1). In addition, a new polyadenylation signal localized on the complementary strand of the 3′ end of the pol gene was proposed [9].

Using constructs harbouring the 3’ LTR positioned in the antisense orientation upstream of the luciferase reporter gene, we also demonstrated that Tat had a positive impact on the antisense promoter activity. However, it is important to note that we have recently demonstrated that Tat-mediated regulation of antisense transcription is rather modest and as such, more experiments will be needed to further evaluate this issue [10].

Using high-throughput sequencing, Lefebvre et al have confirmed the presence of HIV-1 antisense transcripts in SupT1 cells infected with NL4.3ΔenvGFP virions pseudotyped with the VSV-G envelope [11]. In addition, authors showed that antisense transcripts accounted for less than 1% of all viral transcripts. Recently, specific detection of the antisense transcripts in transfected and infected cells were further reported by Kobayashi et al., although differences in transcript length and the position of their extremities were noted in comparison with our findings [12].

In a recent report by Laverdure et al. [13], antisense transcription was shown to be favored in cells of monocytoid origin and confirming the results of Landry et al. [9]. Indeed, Using pNL4.3AsLucE – and pNL4.3LucE- constructs, the ratio between sense and antisense promoter activity was found to be lower in monocyte-derived dendritic cells (MDDCs) than in that of activated CD4+ T lymphocytes [13].

In addition, using fluorescent microscopy analyses it was noted that, when MDDCs express antisense transcripts, cells express very low levels of sense transcripts and the inverse correlation was also confirmed [13]. This last study underscored an important observation, which needs to be further studied in order to better understand the expression pattern of ASP transcript and its derived protein.

Two recent studies have also suggested that HIV-1 antisense ASP-harbouring transcripts could also directly impact viral sense gene expression. In the study by Kobayashi et al. [12], the expression of the antisense transcript was in fact proposed to suppress HIV-1 replication for several weeks.

A study by Saayman et al. further suggested that a long non-coding antisense RNA is produced in infected cells and acts on the promoter activity of the 5’ LTR region by epigenetic silencing, thereby down-modulating viral gene expression [14].

Antisense Protein of HIV-1

Bioinformatic analyses performed by Miller in the first report on ASP revealed that its ORF had the potential to encode a well-conserved protein predicted to be highly hydrophobic [2]. Amino acid distribution analyses revealed that the protein was rich in cysteine, leucine, proline and serine and possessed two potential transmembrane helices with a cytoplasmic N-terminus (Figure 2).

As a result, ASP was predicted to be associated with the endoplasmic reticulum, the plasma membrane and/or embedded in other intracellular membranes [15]. In addition, ASP also displayed a double PxxP motif in its proline-rich hydrophilic region (Figure 2).

Interestingly, this type of motif has been reported to interact with SH3 (SRC Homology 3) binding domains [16, 17].
According to this first study, one major issue was to determine how this antisense ORF is conserved between the different HIV-1 isolates.

Using various alignment softwares, a bioinformatic analysis of 4418 isolates from the Alamos Database (http://www.hiv.lanl.gov) revealed that the ASP ORF is well conserved among different HIV-1 primary isolates [18] (A. Gross, personal communication). Furthermore, the amino terminal extremity is also demonstrated to be highly conserved between the different subtypes of HIV-1 [18]. However, the first 25 amino acids of ASP are absent in subtypes A and A1.

Interestingly, in these 2 subtypes an initiator methionine codon was created (or appeared) at position 26, probably to maintain/conserve a functional ASP ORF (or and preserve a functional ASP ORF) [18]. This suggests that the ASP ORF is conserved during the evolution of HIV-1 and argues
for a role of this protein.

Initial studies allowed the detection of an in vitro translated ASP protein using rabbit sera [19]. Importantly, immunoprecipitation analyses of this protein with sera from HIV-infected patients led to the expected 20-kDa signal, implying a potential correlation between this humoral response and disease progression [19].

Studies by Barbagallo et al., Ludwig et al. and Bansal et al. have also suggested that antisense transcripts also contain several small antisense ORFs, which are potentially synthesized in HIV-1-infected cells and could have the potential to act as cryptic epitopes or modulate ASP expression [8, 20, 21]. Although the ASP protein remains the focus of current studies on HIV-1 viral protein derived from antisense transcripts, these small ORFs also need to be taken into consideration.

Analyses of ASP by using transmission electron microscopy by Vaquero’s group initially demonstrated that this protein could be detected in the cytoplasm of infected cells [15]. However, the protein was also present in intracellular membranes and the plasma membrane and, it was further suggested to be packaged in the virion [15].

As the ASP subcellular localization was only predicted by bioinformatic analyses and analyzed by electron microscopy, we have reassessed its distriburion by confocal microscopy and showed that, in T cell lines, ASP presents a polarized distribution at the plasma membrane [22].

Recently, through the use of codon optimization allowing for better detection of ASP, this protein was readily identifiable by Western blot in cell lines transfected with ASP expression vectors [23]. Furthermore, immunoprecipitation of ASP with anti-Myc antibodies or commercially generated polyclonal anti-ASP antibodies against a conserved peptide (position 47 to 61) was performed and demonstrated a specific signal at the predicted molecular weight of ASP following transfection with ASP expression vectors or with HIV-1 proviral DNA [23].

We also demonstrated that, in addition to its partial localization at the plasma membrane, ASP is a cytoplasmic protein, which is associated with intracellular double- membrane vesicles named autophagosomes, an essential structure for the process of autophagy. Our results further argued that ASP co-localizes and interacts with LC3b-II, a marker of autophagosomes [23].

ASP and autophagy

Other results have further permitted to define a better association between ASP and autophagy.

Indeed, immunoprecipitation of ASP by the anti-ASP polyclonal antibody and Western blot analyses revealed that ASP was
able to form a high molecular weight complex. This finding was further substantiated by a multimerization assay using as cross-linker, glutaraldehyde at different concentrations [23].

From this study, our data have further suggested that the high multimerization potential of ASP is associated with induced autophagy and thereby strongly affects its turnover [23]. Indeed, we have demonstrated that, when ASP was expressed in various cell lines, a typical punctuated distribution became apparent.

Furthermore, this distribution was sensitive to known autophagy inhibitors, 3-methyladenine and bafilomycin A1. Autophagy markers were also monitored to confirm the induction of ASP and we have confirmed this through increased Beclin 1 expression and increase the level of the modified LC3b-II marker, which again were shown to be sensitive to autophagy inhibitors.

Our preliminary results have further demonstrated that cysteine residues and the PxxP motif of ASP are involved in its multimerization and can be linked to its association with autophagosomes (Torresilla et al., unpublished data). However, further studies are needed to understand how ASP can multimerize and how this multimer might impact ASP functions.
Autophagy is a complex cellular process involved in the degradation and recycling of aging organelles and long-lived proteins and is highly relevant in the context of immune responses against various intracellular pathogens [24, 25]. However, viruses can subvert this biological process towards their own advantage [26].

Of interest, this process was recently associated with HIV-1 replication with differences in the outcome depending on the infected cell type [27-36]. Indeed, while the HIV-1 Env protein induces autophagy and apoptosis in neighbouring non-infected CD4+ T cells, in monocytic cells, Kyei et al. have reported that HIV-1 infection stimulates the first steps of autophagy with subsequent increased in HIV-1 replication [33-36].

Their results further suggested an interaction between Nef and Beclin 1, which blocked the final step of autophagy (fusion of autophagosomes with lysosomes ) and thereby, counteracted the proteolytic degradation of HIV-1 virions [33]. In our recent study [23], we have also presented evidence that, in proviral DNA-transfected cells or in infected cells, ASP was important for optimal viral production in several cell lines, including the monocytic U937 cell line.

Importantly, differences in viral production between wild-type and ASP-deficient proviral DNA was greatly diminished when cells were treated with autophagy inhibitors. Based on these results, we thereby propose that one potential function of ASP would be to induce autophagy, which would consequently modulate HIV-1 replication (Figure 3) [23].

Hence, in our model, ASP multimers would induce the formation of the phagophore before or upon binding to LC3b. Upon formation of the mature autophagosomes, entrapped pr55 gag would be more actively processed, which would augment virion production.

Furthermore, the presence of Nef would stabilise these viral proteins by inhibiting late steps of autophagy. This model therefore readdresses previous findings in macrophages [33] and is supported by the fact that the selective impact of ASP in monocytic cells would confirm previous findings showing that natural ASP expression in the proviral DNA context seems more important in infected monocyte-derived cells than in activated CD4+ T cells.

Understanding the role of ASP in autophagy and/or HIV-1 replication in monocyte-derived cells will likely be crucial to achieve a complete understanding of HIV-1 persistence. More studies are needed to confirm this potential role of ASP in autophagy and HIV-1 replication.

Conclusion

In conclusion, the asp gene is expressed through an unspliced transcript and is synthesized as a functional protein with an unusual multimerizing capacity, which, based on our findings, would confer its rapid turnover. Through the formation of multimer, ASP seems to induce autophagy and positively modulate HIV-1 replication in macrophages, probably at a late step of its replication cycle.

It is not excluded that ASP can interact with certain cellular proteins, which could lead to the degradation of these proteins by autophagy but also might act differently in various cellular contexts. Finally, a role of ASP in HIV-1 immune evasion also needs to be studied as autophagy and antigen presentation by MHC class II have been clearly associated with other infections [37].

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Figure 1: Schematic representation of the position of the ASP ORF in HIV-1 proviral DNA. The ASP ORF is localized on the complementary strand of the env gene. Several transcription initiation sites have been localized upstream of the ASP ORF. A consensus polyadenylation signal was identified at a distance of 2.4 kb from the ASP stop codon. The antisense transcript is expected to be approximately 4.1 kb long [9].
Figure 2: Schematic representation of ASP domains. A. The entire amino sequence of ASP is presented above the schematic representation of its various motifs and domains (Genbank accession number: AFK10192.1). B. ASP is a highly hydrophobic protein that is enriched in cysteine, leucine and proline residues. In its N-terminal end, two cysteine triplets are present (CCC) [23]. A double PxxP motif is also present (PxxPxxP) and, based on our unpublished data, might be involved in the capacity of ASP to induce autophagy. The first fifteen amino acids could be involved in ASP multimerization. ASP also harbors two potential transmembrane domains (TM).
Figure 3: Potential role of ASP in autophagy. This model is based on the hypothesis that ASP multimerizes and induces an early step of autophagy [23]. Multimerization of ASP induces autophagy and is then contained in mature autophagosomes. Induced autophagy improves Gag processing and ensuing viral production, mainly in monocytederived cells or monocytic cell lines, which are more prone to favour antisense transcription than activated CD4+T cells [13]. Through its interaction with Beclin 1, Nef would block proteolytic degradation of viral proteins and virions, as previously reported [33].

More information: Elias K. Halvas et al, HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus, Journal of Clinical Investigation (2020). DOI: 10.1172/JCI138099

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