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^pol) 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

Analysis of Gag Polyprotein Processing

<p>Western blot analysis of cell lysates (left panel) or virus particles (right panel) obtained by ultracentrifugation of cell culture supernatants from 293T cells transfected with the respective recombinant virus plasmids in the absence of inhibitor or in the presence of 50 or 500 nM of RO033-4649. Molecular mass standards are depicted on the left, Gag-derived proteins are identified on the right. Protein detection was performed following incubation with a specific antibody against NC.</p