HIV-1 evolution: frustrating therapies, but disclosing molecular mechanisms

Das, Atze T.; Berkhout, Ben

doi:10.1098/rstb.2010.0072

Cited by 16 publications

(10 citation statements)

References 73 publications

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“…These results highlight the many intricacies of these complex virus evolution studies. 45,46 More importantly, we confirm that there is no mechanism of resistance against the combinatorial RNAi therapy of the R3A regimen, which renders this combination truly effective. Based on these promising in vitro results, the therapeutic potential of this combinatorial RNAi approach should be tested in appropriate in vivo models to prepare for a clinical trial in humans.…”

Section: Discussionsupporting

confidence: 63%

The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

2014

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RNA interference (RNAi) is highly effective in inhibiting human immunodeficiency virus type 1 (HIV-1) replication by the expression of antiviral short hairpin RNA (shRNA) in stably transduced T-cell lines. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against highly conserved regions of the HIV-1 RNA genome. The future in vivo application of such a gene therapy protocol will reach only a fraction of the T cells, such that HIV-1 replication will continue in the unmodified T cells, thereby possibly frustrating the therapy by generation of HIV-1 variants that escape from the inhibition imposed by the protected cells. We studied virus inhibition and evolution in pure cultures of shRNA-expressing cells versus mixed cell cultures of protected and unprotected T cells. The addition of the unprotected T cells indeed seems to accelerate HIV-1 evolution and escape from a single shRNA inhibitor. However, expression of three antiviral shRNAs from a single lentiviral vector prevents virus escape even in the presence of unprotected cells. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models.

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Section: Discussionsupporting

confidence: 63%

The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

2014

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“…Second, we anticipated that, should some variants form viable viruses, their passaging in ducks might lead to adaptive mutations that could add to an understanding of the natural conservation versus variability of the Dε sequence, as well the evolutionary capacity of hepadnaviruses in general, an important issue for development of drug resistance and vaccine escape in HBV. Similar in vivo evolution experiments, mostly with bacteriophages or eukaryotic viruses that can be propagated in cell culture, have recently provided surprising new insights into virus evolution (14,15,43,49).…”

Section: Vol 85 2011mentioning

confidence: 99%

A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

Schmid

Rösler

Nassal

2011

J Virol

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In all hepadnaviruses, protein-primed reverse transcription of the pregenomic RNA (pgRNA) is initiated by binding of the viral polymerase, P protein, to the RNA element. Universally, consists of a lower stem and an upper stem, separated by a bulge, and an apical loop. Complex formation triggers pgRNA encapsidation and the -templated synthesis of a DNA oligonucleotide (priming) that serves to generate minus-strand DNA. In vitro systems for duck hepatitis B virus (DHBV) yielded important insights into the priming mechanism, yet their relevance in infection is largely unexplored. Moreover, additional functions encoded in the DHBV (D) sequence could affect in vivo fitness. We therefore assessed the in vivo performances of five recombinant DHBVs bearing multiple mutations in the upper D stem. Three variants with only modestly reduced in vitro replication competence established chronic infection in ducks. From one variant but not another, three adapted new variants emerged upon passaging, as demonstrated by increased relative fitness in coinfections with wild-type DHBV. All three showed enhanced priming and replication competence in vitro, and in one, DHBV e antigen (DHBeAg) production was restored. Pronounced impacts on other D functions were not detected; however, gradual, synergistic contributions to overall performance are suggested by the fact of none of the variants reaching the in vivo fitness of wild-type virus. These data shed more light on the P-D interaction, define important criteria for the design of future in vivo evolution experiments, and suggest that the upper D stem sequences provided an evolutionary playground for DHBV to optimize in vivo fitness.Hepadnaviruses are small enveloped hepatotropic DNA viruses that replicate through protein-primed reverse transcription (for reviews, see references 8 and 38). Human hepatitis B virus (HBV) and duck HBV (DHBV) are the respective prototypes of the mammalian and avian HBVs, which share a similar genome organization and replication strategy (Fig. 1A). Their ϳ3-kb genomes encode a single core protein, three or two (avian viruses) envelope proteins, an unusual reverse transcriptase (P protein), and, only in the mammalian viruses, the poorly understood X protein. Virions contain the genome as a relaxed circular DNA (rcDNA), which upon infection is converted into nuclear covalently closed circular DNA (cccDNA), the template for all viral transcripts. In addition to the subgenomic RNAs (sgRNAs), two types of greater-than-genomelength RNAs are produced. The precore RNAs serve for translation of the precursors of the secretory hepatitis B virus e antigens (HBeAg and DHBeAg, respectively), which may modulate the host's immune response but are nonessential. The pregenomic RNAs (pgRNAs), by contrast, are vital as mRNAs for core and P protein and by constituting the obligate template for new DNA genomes.Reverse transcription begins with specific packaging of pgRNA into newly forming capsids, triggered by binding of P protein to the 5Ј-proximal ε (Dε for DHBV) stem-loop (...

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“…As input for virus selection, we used a virus mixture of wt-D30N-L90M at the ratio of 10:1:1. This makes the virus evolution experiment independent of the initial mutation step, which otherwise would create much experimental variation, as mutation is a chance process (3,10). SupT1 cells expressing second-generation shRNA-D30N, -L90M, or -combi (D30N and L90M) were infected with this virus mixture, and cells expressing no shRNA (JS1) served as a control.…”

Section: Resultsmentioning

confidence: 99%

Directed HIV-1 Evolution of Protease Inhibitor Resistance by Second-Generation Short Hairpin RNAs

Schopman

Braun

Berkhout

2012

Antimicrob Agents Chemother

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Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PIresistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches. Worldwide, more than 35 million individuals are infected with human immunodeficiency virus type 1 (HIV-1), and each year approximately 3 million persons are newly infected. Despite the major success of antiretroviral therapy (ART) that delays or prevents the onset of disease, HIV-1 infections remain incurable, and attempts to make a vaccine have so far proved unsuccessful (14). The emergence of drug-resistant viruses remains a major problem for some patients, especially those who exhibit suboptimal therapy adherence.The RNA interference (RNAi) mechanism is a posttranscriptional gene silencing process that holds promise as a new antiviral strategy. RNAi is induced by double-stranded RNA (dsRNA) that is processed by the RNAi machinery into small interfering RNAs (siRNAs). The siRNAs are designed with perfect base pairing complementarity to the target RNA sequence and trigger cleavage of the targeted mRNA (5, 8). HIV-1 can be inhibited effectively and specifically by RNAi in vitro. Many HIV-1 genes have been targeted by transfected siRNAs or intracellular expressed short hairpin RNAs (shRNAs), and combinatorial RNAi strategies can durably inhibit HIV-1 replication (2,11,31). Antiviral drugs and siRNAs can also be combined (9). One successful RNAi-based approach concerns the use of second-generation shRNAs designed to target the favorite escape variants that are selected under pressure of first-generation shRNAs, thus skewing virus evolution (23). In this study, we designed second-generation shRNAs to counter the evolution of clinically relevant drug-resistant HIV-1 variants.We investigated the potential of combining two anti-HIV strategies. Protease inhibitors (PIs) that successfully suppress HIV-1 replication were combined with second-generation shRNAs to block the favorite viral escape routes. To do so, we first designed second-generation shRNAs and tested them in repo...

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HIV-1 evolution: frustrating therapies, but disclosing molecular mechanisms

Cited by 16 publications

References 73 publications

The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

Directed HIV-1 Evolution of Protease Inhibitor Resistance by Second-Generation Short Hairpin RNAs

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