Assembly of a functional Machupo virus polymerase complex.
Segmented negative-sense viruses of the family Arenaviridae encode a large polymerase (L) protein that contains all of the enzymatic activities required for RNA synthesis. These activities include an RNA-dependent RNA polymerase (RdRP) and an RNA endonuclease that cleaves capped primers from cellular mRNAs to prime transcription. Using purified catalytically active Machupo virus L, we provide a view of the overall architecture of this multifunctional polymerase and reconstitute complex formation with an RNA template in vitro. The L protein contains a central ring domain that is similar in appearance to the RdRP of dsRNA viruses and multiple accessory appendages that may be responsible for 5' cap formation. RNA template recognition by L requires a sequence-specific motif located at positions 2-5 in the 3' terminus of the viral genome. Moreover, L-RNA complex formation depends on single-stranded RNA, indicating that inter-termini dsRNA interactions must be partially broken for complex assembly to occur. Our results provide a model for arenavirus polymerase-template interactions and reveal the structural organization of a negative-strand RNA virus L protein.
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Philip J
PJ
Kranzusch
Andreas D
AD
Schenk
Amal A
AA
Rahmeh
Sheli R
SR
Radoshitzky
Sina
S
Bavari
Thomas
T
Walz
Sean P J
SP
Whelan
D006477:Arenaviruses, New World / Q000201:enzymology*; D001483:Base Sequence; D055162:Biocatalysis; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D013698:Templates, Genetic; D014764:Viral Proteins / Q000302:isolation & purification / Q000648:ultrastructure
D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase