Fig 3. Production of replication-defective SIV particles by rRRV-SIV-nfl.

A) Early passage rhesus fibroblast (RF) cells were grown in 6-well culture plates and subsequently infected with 3.5x10⁹ genome copies of the indicated recombinant SIV-nfl. The RF cell culture supernatants were collected at the indicated time points post infection and subjected to an antigen capture assay to measure the levels of SIV Gag p27. Supernatant of uninfected RF cells served as a negative control. B) Cell culture supernatant was harvested from early passage rhesus fibroblast (RF) cells infected with the respective recombinant RRV-SIV-nfl strains. Subsequently, CEMx174 cells were infected with these supernatants containing 20ng of p27 as measured by an antigen capture assay. An equal amount (20ng of p27) of SIVmac239 wild-type virus produced in HEK239T-cells served as a positive control. The supernatants from the CEMx174 cell cultures were collected at the indicated days post infection and were used in an antigen capture assays measuring p27 levels. Supernatant of uninfected RF cells served as a negative control. C) Early passage rhesus fibroblast (RF) cells grown in T75 culture flasks were infected with rRRV-SIVcmv-nfl and harvested at days 4–6 post infection. Subsequently, the cells were fixed with 2.5% glutaraldehyde in sodium cacodylate buffer. Transmission electron microscopic (TEM) images were taken at The Core Electron Microscopy Facility, University of Massachusetts. Shown are SIV particles budding from cell membrane (filled arrowheads). The bar in the lower left corner represents 500 nm D) TEM image showing free SIV particles including mature SIV particles (filled arrowheads) containing cylindrical, rod-shaped nucleoid as well as immature particles lacking any nucleoid structures (empty arrowheads). The bar in the lower left corner represents 200 nm. In the inset on the upper right, a TEM image of recombinant RRV derived from a separate image is shown.