Replication of flaviviruses: Separation of membrane translation sites of kunjin virus proteins and of cell proteins

Abstract

Infected Vero cells were labeled with radioactive amino acids, mannose, or uridine during the peak period of virus production. By using mild cell disruption and preparative procedures, three cell extracts were obtained: a cytoplasmic extract (1), membranes mechanically stripped from nuclear pellets (2), and material extracted with double detergent from the stripped nuclei (3). Labeled heavy (H), intermediate (I), and light (L) density fractions coincident with visible bands of membranes were obtained from the extracts, by sedimentation for 3 hr through a discontinuous sucrose gradient. After a 10-min pulse of [ 3H]uridine, two-thirds of the incorporation was in Extract 1 where it was located in the light membranes (i.e., in L-1) and was 90% RNase resistant in situ. After a 3-hr labeling period, [ 3H]uridine was distributed also through the denser fractions of Extract 1, but the RNase resistance in L-1 was then only 16%; [ 3H]uridine accumulated also in H-2 (heavy membranes of Extract 2), and in a dense or polysome-like fraction of Extract 3. Intracellular and extracellular progeny virions cosedimented with L-1. Pulse-chase experiments showed that the heavy membranes H-1 and H-2 were the site of translation and of glycosylation of structural and nonstructural virus-specified proteins, whereas the L-1 fraction was the major site of translation of cell proteins in infected and in mock-infected cells. During a 10-min pulse with [ 3H]mannose, the virus-specified glycoproteins gp66, gp59.5, and NV2 (a nonstructural protein, molecular weight 19,000) were labeled, and during a 1-hr chase label accumulated in gp53.5 at the expense of gp66. No amino acid-labeled polypeptides corresponding to gp66 and gp53.5, both of which appear to be related to gp59.5 and to the envelope protein V3 (molecular weight 51,300), were seen in electropherograms. Transfer occurred during the 1-hr chase of several virus-specified proteins and of the glycoproteins gp59 and gp53.5, mainly into the I-1 and L-3 fractions, but most translation products remained in H-1 and H-2. The translation and glycosylation of flavivirus proteins (structural and nonstructural) appear to be unique in that these processes are completed at a specific cell site (heavy membranes), separable from the sites of concurrent cell protein translation and of virus transcription (predominantly in the L-1 fraction).