11 research outputs found
A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism
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
Intensive Care Admissions for Severe Chikungunya Virus Infection, French Polynesia
During the 2014–2015 chikungunya outbreak in French Polynesia, 64 patients with confirmed chikungunya virus infection were admitted into intensive care. Sixty-three were nonpregnant adults; 11 had an atypical form, 21 had severe sepsis or septic shock, and 18 died. These findings indicate that critical illness frequently complicates the course of chikungunya virus infection
Co-evolution of nelfinavir-resistant HIV-1 protease and the p1-p6 substrate
The selective pressure of the competitive protease inhibitors causes both HIV-1 protease and occasionally its substrates to evolve drug resistance. We hypothesize that this occurs particularly in substrates that protrude beyond the substrate envelope and contact residues that mutate in response to a particular protease inhibitor. To validate this hypothesis, we analyzed substrate and protease sequences for covariation. Using the chi2 test, we show a positive correlation between the nelfinavir-resistant D30N/N88D protease mutations and mutations at the p1-p6 cleavage site as compared to the other cleavage sites. Both nelfinavir and the substrate p1-p6 protrude beyond the substrate envelope and contact residue 30, thus possibly making the p1-p6 cleavage site more vulnerable to co-evolution
Quantification of Human Cytomegalovirus DNA in Bone Marrow Transplant Recipients by Real-Time PCR
A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Unlike other teams, we quantified CMV and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using a plasmid containing both sequences as an external standard. Tenfold serial dilutions of this plasmid yielded overlapping standard curves that allowed the quantification of CMV and GAPDH gene copies in an efficient and accurate manner. Sequential blood samples (164 specimens) were collected from 16 patients. PBLs were tested by the pp65 antigenemia assay and quantitative CMV and GAPDH gene PCRs. CMV DNA was detected by PCR in 13 patients a mean of 15 days prior to the appearance of antigenemia. The administration of anti-CMV drugs led to a rapid decrease in the numbers of viral copies and positive nuclei. Real-time PCR assay results correlated with those of the CMV pp65 antigenemia assay (P < 0.00001). The TaqMan assay may be a useful tool for rapid quantification of CMV infection and for monitoring of CMV reactivation in bone marrow transplantation recipients
Schematic Representation of the Viral Frameshift Region and Investigation of Frameshift Efficiency
<div><p>The RNA structure of the frameshift stimulatory signal is represented here [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040036#pmed-0040036-b030" target="_blank">30</a>,<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040036#pmed-0040036-b051" target="_blank">51</a>]. The nucleotide and amino acid changes (between brackets) as observed in our in vitro selection experiments are indicated.</p>
<p>(A) The relative frameshift efficiency of wild-type HIV (HXB2) and the p1 constructs of IVS-1 (436E+437T), IVS-32 (437V), and IVS-34 (437T). The values of the frameshift efficiency are the means of four independent experiments, with bars representing the standard errors.</p>
<p>(B) Analysis of differences in levels of HIV GagPol products (RT, p66, and p51) as compared to HIV Gag products (CA, p24) by Western blot analysis.</p></div
Determination of Phenotypic Drug Susceptibility of C-Terminal Gag Clones
<p>Representation of the fold increases in phenotypic drug susceptibility of the C-terminal Gag clone derived from IVS-1, containing the 436E+437T amino acid change as compared to wild-type HIV (HXB2). The black bars indicate the fold increase as determined in the multiple-cycle MTT drug susceptibility assay, and the gray bars indicate the fold increase as determined in the single-cycle PhenoSense drug susceptibility assay. Drug susceptibility to the PI RO033-4649, LPV, TPV, ATV, AMP, ritonavir (RTV), saquinavir (SQV), indinavir (IDV), and nelfinavir (NFV), and as a control to the RT inhibitor NVP was determined. RO033-4649 and TPV were not available at the time of PhenoSense assay testing.</p
Schematic Representation of the Distribution of all Amino Acid Changes Appearing during in vitro Selection Experiments Using RO033-4649
<p>Horizontal bars below the gene organization scheme represent the genomes of viruses from the three in vitro selection experiments: IVS-1, IVS-32, and IVS-34. Vertical lines illustrate the observed amino acid changes. The C-terminal portion of Gag was expanded to precisely map the nucleotide changes leading to amino acid changes (bold) in both translational reading frames (Gag and GagPol). In addition, the three different cleavage sites (NC/p1, NC/TFP, and TFP/p6<sup>pol</sup>) are indicated. The control experiments, in which no drug was added during the in vitro selections, demonstrated no amino acid changes in the viral Gag and protease region.</p