FIG. 2.
Establishment of a highly sensitive and specific rat integration PCR and of basic pharmacovirological parameters of GSK501015 in rats. (A) Strategy of the “optimized protocol” for the nested rat integration PCR (see Materials and Methods for details). ID, rodent identifier consensus sequence. (B) The sensitivities of detection of HIV-1 integrations in the rat genome in a recently reported nested PCR protocol (standard protocol [19]) and the optimized protocol developed here were compared. Shown are the relative intensities of the specific signals (left), the percentage of unspecific signal (middle), and the lower limit of the standard (right), applying the two PCR protocols to the Rat2int genomic standard. (C) GSK501015 potently suppresses HIV-1 integrant formation in a rat cell line. Rat2 cells were pretreated with different concentrations of GSK501015 in the presence (1 μM) (negative control) or absence of the RTI efavirenz and challenged with a VSV-G-pseudotyped HIV-1 GFP vector. On day 7 postinfection, DNA was extracted from passaged cells, and the relative levels of HIV-1 integrants were determined as described in Materials and Methods. The number of integrants in the absence of treatment was set to 100%, and the relative percentages in the presence of decreasing concentrations of GSK501015 are depicted. (D) Relationship of oral dose and serum concentration of suspension-formulated GSK501015 in outbred, nontransgenic Sprague-Dawley rats. Data points indicate arithmetic means ± standard deviations (n = 3 animals).