Asymmetric packaging of polymerases within vesicular stomatitis virus

•The VSV polymerases (L proteins) are localized to the blunt end of the virus.•The VSV phosphoproteins (P proteins) are localized to the blunt end of the virus.•Each VSV virion packages a variable number of P and L proteins. Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57±12nm of the 150nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.