Fig. 4.

Synthetic viral nanosystems. Synthetic viral nanosystems (SVNs) are created by customizing MultiBac baculoviral genomes through integration of additional functionalities into the viral LoxP site. Selected examples are depicted in the box on the right (panels a–c). a TheMultiBac-based “kinase factory” contains seven chaperones that assist in the folding of kinase enzymes, thus enhancing the stability and yield of functional protein. Coomassie-stained SDS-PAGE sections show test expression of KRS1, with the MultiBac virus (left lanes) and the kinase factory (right lanes), respectively. T total cell extract, S soluble protein. The asterisk denotes KRS1, which was expressed as a glutathion-S-transferase (GST) fusion protein. Molecular weight marker band sizes (in kDa) are indicated by numbers. The kinase is barely visible in the MultiBac expressions. By contrast, high-level expression of kinase is observed with the kinase factory. b “Virus-like particle (VLP) factory” was constructed by integrating the gene encoding influenza H1N1 M1 capsid protein into the viral LoxP site, together with the florescent protein mCherry-encoding gene to track virus performance. The VLP factory efficiently produces influenza virus-like particles decorated with envelope proteins co-expressed from the viral Tn7 site. VLP is shown in a schematic drawing (courtesy of D. Jordan), with the capsid formed by M1 protein colored in brown. The VLP is devoid of genetic content but displays hemagglutinin (blue spikes) and neuraminidase (purple spikes) on its surface in a live-virus-like fashion, embedded in a host-cell derived lipid bilayer envelope. An EM image of influenza VLP produced by using VLP factory is shown beneath the schematic. c The MultiBacTAG SVN. Orthogonal pyrolysyl tRNA (tRNA ^Pyl) and cognate synthetase (PylRS) from Methanosarcina mazei inserted in the viral LoxP site enable integration of artificial (non-canonical) amino acids (ncAA) by UAG stop-codon suppression. The growing polypeptide chain contains the artificial amino acid (ncAA, red star) at the position determined by the UAG codon. MultiBacTAG was used to integrate a UV-activatable amino acid into a protein complex formed by human transcription factors TAF11 and TAF13, revealing specific crosslinks in the presence of TBP (drawn schematically in gray). A section from a western blot stained with anti-TAF13 antibody shows the cross-link upon UV radiation. Reprinted by permission from Macmillan Publishers Ltd: Nature Methods 13:997-1000, copyright 2016 [58]