BROME MOSAIC-VIRUS HELICASE-LIKE AND POLYMERASE-LIKE PROTEINS COLOCALIZE ON THE ENDOPLASMIC-RETICULUM AT SITES OF VIRAL-RNA SYNTHESIS

The helicase-like 1a and polymerase-like 2a proteins of brome mosaic v irus (BMV) are required for viral RNA replication in vivo, are present in membrane-bound viral RNA polymerase extracts, and share conservati on with the many other members of the alphavirus-like superfamily. To better understand BMV RNA replication and BMV-host interactions, we us ed confocal microscopy and double-label immunofluorescence to determin e and compare the sites of la, 2a, and nascent viral RNA accumulation in Bhn-infected barley protoplasts. la and 2a showed nearly complete c olocalization throughout infection, accumulating in defined cytoplasmi c spots usually adjacent to or surrounding the nucleus, These spots gr ew throughout infection and by 16 h postinoculation often assumed a ve sicle like appearance. The BMV RNA replication complex incorporated 5- bromouridine 5'-triphosphate into RNA in vitro and in vivo, allowing i mmunofluorescent detection of nascent RNA. The cytoplasmic sites of BM V-specific RNA synthesis coincided with the sites of 1a and 2a accumul ation, and at the resolution of confocal microscopy, all sites of la a nd 2a accumulation were sites of BMV RNA synthesis. Double-label immun ofluorescence detection of selected subcellular markers and la or 2a s howed that BMV replication complexes were tightly associated with mark ers for the endoplasmic reticulum but not the medial Golgi or later co mpartments of the cellular secretory pathway. Defining this associatio n of BMV RNA replication complexes with endoplasmic reticulum markers should assist in identifying and characterizing host factors involved in BMV RNA replication.