Synthesis of template-sense, single-strand flockhouse virus RNA in a cell-free replication system

Abstract

Cell-free extracts of Drosophila melanogaster cells infected with FHV (Flockhouse virus) have the capacity to synthesize FHV RNA. When such extracts are incubated appropriately, ss and ds FHV RNA 1, -2, and -3 can be detected as synthesis products. Pulse-chase analyses indicate that radioactivity, incorporated into ds RNA 1, -2, and -3 is chased into ss RNA 1, -2, and -3, suggesting that these ds RNAs are intermediates in FHV RNA replication. When the cell-free extracts are treated with detergents to solubilize cellular membranes, only ds RNA 1, -2, and -3 can be detected as synthesis products. The cell-free extracts, treated with a ribonuclease to remove endogenous templates, have the capacity to replicate FHV RNA 1 and -2 when these RNAs are provided as templates, together with lipofectin. Both as and ds RNA 1 and -2 are detected as synthesis products. When RNA3 is provided as a template, only ds RNA3 is synthesized. In the absence of lipofectin or in the presence of detergent, only ds RNAs are detected as synthesis products and all the incorporated radioactivity is found in the strand complementary to that of the template. Our results are consistent with a mechanism in which second-strand synthesis, although not complementary strand synthesis, requires intact membranes. They also suggest that ds RNA3 is an intermediate in the synthesis of ss RNA3.