The Major Cellular Sterol Regulatory Pathway Is Required for Andes Virus Infection
Figure 5
Quantitative PCR analysis of viral RNA during infection of human cells.
(A) HAP1WT or HAP1S1P cells were infected with VSV-(G) or rVSV-ANDV for 12 hours and cells collected for flow cytometric analysis. Infection was normalized to infection levels in HAP1WT cells. Mean±SEM is shown for five independent experiments; ** p<0.01. (Average raw infection percentages (HAP1 WT): VSV-(G) = 55%, rVSV-ANDV = 71%) (B) Binding and internalization of rVSV-ANDV. rVSV-ANDV was bound to three sets of HAP1WT and HAP1S1P cells for one hour on ice. One set of cells were scraped into PBS and washed to measure bound virions (bound). A second set of cells were treated with trypsin for 10 minutes to remove externally bound virions (background). A third set of cells were warmed to 37°C for one hour to permit endocytosis before being treated with trypsin to remove any remaining external virions (internal). Cells were washed extensively, cell pellets and associated virions were lysed for RNA extraction and viral RNA (vRNA) was quantified by qRT-PCR. Viral RNA values were normalized to GAPDH to control for input RNA levels and plotted relative to virus bound to HAP1WT cells. Mean±SEM is shown for three independent experiments; ** p<0.01, ***p<0.001.