Figure 2.
HIV Incorporates IP5 in the Absence of IP6 without Loss of Production or Infectivity
(A) TiO2-PAGE and toluidine blue staining of cell extracts showing IP5 and IP6 levels in IPPK CRISPR/Cas9 knockout clones.
(B) Inositol phosphate quantification in selected IPPK-KO clones using 3H-inositol labeling and inositol phosphate fractionation by SAX-HPLC.
(C) Quantification of IP5 and IP6 packaging in virions produced in wild-type and IPPK-KO cells through [3H]inositol labeling, SAX-HPLC, and scintillation counting of fractions.
(D) p24 western blot of pelleted virions showing p24 levels in HIV virions produced from IPPK-KO clones.
(E) Measurement of virus production through quantification of RT in viral supernatants from IPMK-KO clones. Error bars depict mean ± SD of three independent experiments. Values are represented as fold WT virus, and reduction compared with WT is statistically significant (p < 0.0012 in all cases).
(F) Infectivity of viruses from (E), as a function of viral dose measured by RT levels. Error bars depict mean ± SD of three replicates from one experiment representative of three independent experiments.
(G) Membrane flotation analysis of cell lysates from WT, IPMK-KO, and IPPK-KO cells. Western blotting of sucrose gradient fractions for Gag show that similar levels of Gag are associated with the membrane fractions. Gag precursor Pr55Gag (pr55), p41, and mature capsid protein (p24) are indicated.
(H) Virus release assays showing levels of Gag in lysates and virions after transduction of WT and KO cells with virus produced in WT cells. Graph shows relative quantification of p24 from two independent experiments and western blots. Representative data from at least two independent experiments are shown, unless otherwise indicated. CPM data are normalized to background.