MDMs infected by HIV activate STAT1 by means of either

nef or env expression. (A) FACS analysis for the expression of HIV-1 in MDMs 24 hours after challenge. Peripheral blood human MDMs from healthy donors were infected with 10 ng p24 Gag/10⁶ cells of different VSV-G pseudotyped HIV-1 strains, and the percentages of cells expressing HIV-1 were scored 24 hours afterward through intracytoplasmic FACS analysis performed by using a PE-conjugated anti-Gag HIV-1 mAb on cells infected with full-length (fl) HIV-1, or with env,nef, or env/nef deletion mutants. As controls, uninfected cells were labeled with either isotype-matched, unspecific PE-conjugated immunoglobulins (lane a) or with anti-Gag HIV-1 mAbs (lane b). (B) Western blot analysis of phosphotyrosine-STAT1, STAT1, and β-tubulin levels performed on total cell extracts assayed at different times after infection with VSV-G pseudotyped full-length HIV-1 strain. Cell extracts analyzed (A,B) were obtained by pooling simultaneous cell cultures from 4 healthy donors each.(C) Phosphotyrosine-STAT1, STAT1, and β-tubulin levels assayed through Western blots performed on total extracts of cells harvested 8 hours after infection with indicated VSV-G pseudotyped HIV-1 and indicated mutants thereof. Analyses were performed on cell extracts obtained by pooling simultaneous cell cultures from 4 healthy donors. In both panels B and C, specific signals are marked on the left side; kilodaltons of molecular size markers are reported on the right.