Ultrastructural characterization of HISV-1 virions.

A) Analysis of fractions from sucrose density gradient ultracentrifugation of HISV-1 by SDS-PAGE, the proteins were visualized using Coomassie staining. The fractions were collected from the bottom of the gradient (from higher to lower density). The fractions F6-F8 contain the majority HISV-1 as judged by the intensity of ~70 kDa NP band, these fractions were pooled, concentrated and analyzed by WB (shown in panel B) to confirm the presence of NP. The molecular weight marker (Precision Plus Protein All Blue Prestained Protein Standards, Bio-Rad) is at leftmost lane. The figure was obtained using Odyssey infrared imaging system (LI-COR). B) The panel on right shows comparison of the proteins in purified preparation of UGV-1 (reptarenavirus) and HISV-1 (hartmanivirus), as seen by Coomassie staining. The arrows indicate the viral proteins as identified by mobility in SDS-PAGE, the label GP1 and GP2 shows the region where the processed GP1 and GP2 would likely migrate. The molecular weight marker (Precision Plus Protein All Blue Prestained Protein Standards, Bio-Rad) is at leftmost lane. The panels on right show Ponceau S (total protein) staining and WB of purified HISV-1 (pooled fractions F6-F8, panel A) and UGV-1 with anti-HISV NP antiserum. Ponceau S staining was recorded with regular flatbed scanner, the Coomassie stained gel and WB were obtained using Odyssey infrared imaging system (LI-COR). C) Top panel shows HISV-1 examples of virions in EM under negative staining, the scale bar is 100 nm. The bottom panel shows HISV-1 and UGV-1 as seen in cryoelectron microscopy, the scale bar is 200 nm.