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      A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

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          Abstract

          Background

          HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors.

          Methods and Findings

          We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy.

          Conclusions

          HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

          Abstract

          Changes in the cleavage site of the Gag substrate for the HIV protease can convey resistance to protease inhibitors and might contribute to virologic failure during therapy that includes these drugs.

          Editors' Summary

          Background.

          Twenty-five years ago, infection with the human immunodeficiency virus (HIV)—the causative agent of AIDS—was a death sentence. However, drugs that attack various stages of the HIV life cycle were soon developed that, although not curing the infection, kept it in check when used in combination and greatly increased the life expectancy of people infected with HIV. Unfortunately, viruses resistant to these drugs have rapidly emerged and antiviral therapy now fails in many patients. The use of HIV protease inhibitors (PIs) in combination therapies, for example, has led to the stepwise selection of viral variants resistant to these drugs. Resistance is first acquired when the viral protease changes so that PIs no longer bind to it and inhibit it efficiently. These changes often reduce the efficiency with which the protease binds its substrates—polyproteins called Gag and GagPol that it chops up into smaller proteins to make new viral particles. So the next step is the accumulation of changes elsewhere in the protease that make it work better, and sometimes changes in its substrate that make it easier to cut; these compensatory changes do not directly affect viral resistance to PIs.

          Why Was This Study Done?

          To prevent viruses with resistance to PIs emerging, drug doses are kept high in patients and new PIs are being developed with high potency against known PI-resistant HIV variants. Both approaches set a “high genetic barrier” to the development of PI resistance by ensuring that HIV has to incorporate many changes in its protease to become resistant. But, the HIV genome naturally changes—mutates—very rapidly, so novel HIV variants could emerge that are less susceptible to the new potent PIs without the virus having to leap this high genetic barrier. In this study, the researchers have investigated whether HIV can find an alternative route to PI resistance that does not involve the introduction of multiple changes into its protease.

          What Did the Researchers Do and Find?

          The researchers took wild-type HIV and treated it in the laboratory with a new PI regimen that has a high genetic barrier. By gradually increasing its concentration, the researchers selected three viral populations that were able to grow in 4- to 8-fold higher concentrations of the PI than wild-type virus. None of these populations had mutations in the viral protease. Instead, they all had mutations near one of the sites—the NC/p1 site—where the protease normally cuts the Gag polyprotein. These mutations, the researchers report, enhanced the overall efficiency with which the wild-type protease cleaved the polyprotein, and a selection experiment with another PI showed that the development of PI resistance through alterations near the NC/p1 cleavage site was not unique to one PI. The researchers also investigated the potential clinical significance of this new drug resistance mechanism by looking for the same mutations in nearly 30,000 patient samples. Many of the samples did indeed have these mutations. Finally, they showed that mutations at the NC/p1 cleavage site were associated with virological failure (increased viral replication) during PI therapy in an ongoing clinical trial.

          What Do These Findings Mean?

          These results suggest that increased polyprotein processing because of mutations in the natural substrate of the HIV protease might be a new mechanism by which HIV can become resistant to PIs. This strategy, which occurs in the laboratory and in patients, allows HIV to develop PI resistance without the need for multiple changes in its protease and so avoids the high genetic barrier to resistance that new PIs provide. Clinical studies are now needed to test which of the mutations seen in this study contribute to virological failure, whether the degree of this failure is clinically relevant, and whether these substrate mutations enhance the effect of protease mutations. If the clinical importance of the new mechanism is confirmed, genetic examination of both the polyprotein and the protease will be needed when trying to figure out why a PI-containing therapy is failing in individual patients. Furthermore, it will be necessary to test whether this mechanism can contribute to the development of resistance when evaluating new drugs.

          Additional Information.

          Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040036.

          Related collections

          Most cited references52

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          Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation.

          DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes. The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as well. An example for this is the finding that almost all of the soluble DNA in the reaction mix can be bound into an insoluble complex with calcium phosphate in <1 min. Extending the reaction time to 20 min results in aggregation and/or growth of particles and reduces the level of expression. With improved protocols we gained better reproducibility and higher efficiencies both for transient and for stable transfections. Up to 60% of cells stained positive for beta-gal and transient production of secreted proteins was improved 5- to 10-fold over results seen with transfections using standard procedures. Similar improvements in efficiency (number of recombinant cell colonies) were observed with stable transfections, using co-transfected marker plasmids for selection. Transient expression levels 2 days after DNA transfer and titers obtained from stable cell lines, emerging weeks later, showed strong correlation.
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            A dual-luciferase reporter system for studying recoding signals.

            A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.
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              In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors.

              Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease have entered clinical study as potential therapeutic agents for HIV-1 infection. The clinical efficacy of HIV-1 reverse transcriptase inhibitors has been limited by the emergence of resistant viral variants. Similarly, variants expressing resistance to protease inhibitors have been derived in cell culture. We now report the characterization of resistant variants isolated from patients undergoing therapy with the protease inhibitor MK-639 (formerly designated L-735,524). Five of these variants, isolated from four patients, exhibited cross-resistance to all members of a panel of six structurally diverse protease inhibitors. This suggests that combination therapy with multiple protease inhibitors may not prevent loss of antiviral activity resulting from resistance selection. In addition, previous therapy with one compound may abrogate the benefit of subsequent treatment with a second inhibitor.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Med
                pmed
                PLoS Medicine
                Public Library of Science (San Francisco, USA )
                1549-1277
                1549-1676
                January 2007
                16 January 2007
                : 4
                : 1
                : e36
                Affiliations
                [1 ] Eijkman Winkler Center, Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, Netherlands
                [2 ] APHP-Hopital Bichat, Laboratoire Virologie, Paris, France
                [3 ] Monogram Biosciences, San Francisco, California, United States of America
                [4 ] Department of Virology, University of Heidelberg, Heidelberg, Germany
                [5 ] Institute of Organic Chemistry and Biochemistry, Academy of Science of the Czech Republic, Prague, Czech Republic
                [6 ] Roche Bioscience, Palo Alto, California, United States of America
                [7 ] Department of Biochemistry, Université de Montreal, Montreal, Canada
                McGill University AIDS Centre, Canada
                Author notes
                * To whom correspondence should be addressed. E-mail: c.boucher@ 123456umcutrecht.nl
                Article
                06-PLME-RA-0115R3 plme-04-01-12
                10.1371/journal.pmed.0040036
                1769415
                17227139
                d286b31a-a617-4ec0-a665-07a89964e422
                Copyright: © 2007 Nijhuis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 8 February 2006
                : 31 October 2006
                Page count
                Pages: 12
                Categories
                Research Article
                Biochemistry
                Biochemistry
                Infectious Diseases
                Infectious Diseases
                Microbiology
                Virology
                HIV Infection/AIDS
                Drugs and Adverse Drug Reactions
                Chronic Disease Management
                Microbiology
                Custom metadata
                Nijhuis M, van Maarseveen NM, Lastere S, Schipper P, Coakley E, et al. (2007) A novel substrate-based HIV-1 protease inhibitor drug resistance mechanism. PLoS Med 4(1): e36. doi: 10.1371/journal.pmed.0040036

                Medicine
                Medicine

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