3 OF 4 NUCLEOCAPSID PROTEINS OF MARBURG-VIRUS, NP, VP35, AND L, ARE SUFFICIENT TO MEDIATE REPLICATION AND TRANSCRIPTION OF MARBURG VIRUS-SPECIFIC MONOCISTRONIC MINIGENOMES

This paper describes the first reconstituted replication system establ ished for a member of the Filoviridae, Marburg virus (MBGV). MBGV mini genomes containing the leader and trailer regions of the MBGV genome a nd the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsid ated and could he passaged. Unlike most other members of the order Mon onegavirales, filoviruses possess four proteins presumed to be compone nts of the nucleocapsid (NP, VP35, VP30, and L). To determine the prot ein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 express ion system. Northern blot analysis of viral RNA revealed that three nu cleocapsid proteins (NP, VP35, and L) were essential and sufficient fo r transcription as well as replication and encapsidation, These data i ndicate that VP35, rather than VP30, is the functional homologue of rh abdo- and paramyxovirus P proteins, The reconstituted replication syst em was profoundly affected by the NP-to-VP35 expression ratio. To inve stigate whether CAT gene expression was achieved entirely by mRNA or i n part by full-length plus-strand minigenomes, a copy-back minireplico n containing the CAT gene but lacking MBGV-specific transcriptional st art sites was employed in the artificial replication system. This cons truct was replicated without accompanying CAT activity, It was conclud ed that the CAT activity reflected MBGV-specific transcription and not replication.