Protein requirements for assembly of virus-like particles of Junonia coenia densovirus in insect cells

The coding sequences of four overlapping polypeptides starting at four diff erent in-frame AUG codons and co-terminating at the stop codon of the cap g ene of Junonia coenia densovirus (JcDNV) were inserted under the control of the p10 promoter of Autographa californica nucleopolyhedrovirus (AcMNPV) t o generate AcMNPV-VP1 (four polypeptides), AcMNPV-VP2 (three polypeptides), AcMNPV-VP3 (two polypeptides), and AcMNPV-VP4 (one polypeptide) recombinan t viruses. In all cases, infection of Spodoptera frugiperda cells (Sf9) by each of the four recombinant viruses resulted in the production of virus-li ke particles (VLPs) 22-25 nm in diameter, The VLPs produced by the three re combinants AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-VP4 were abundant and containe d three, two and one polypeptides, respectively. VP4, the shortest polypept ide, thus appears to be sufficient for assembly of VLPs morphologically sim ilar to those formed with two to four polypeptides. The ratio of VPs did no t appear to be critical for assembly of the particles. The polypeptide star ting at the first AUG immediately downstream from the p10 promoter was alwa ys the most abundantly expressed in infected cells, regardless of the const ruct. In contrast, plaque-purified AcMNPV-VP1 recombinants were unstable an d produced less than one-twentieth of the VLPs produced by the others. All VP transcripts started at the TAAG late motif of the p10 promoter and had a poly(A) tail 14 nt downstream of a poly(A) addition signal located 98 nucl eotides downstream of the common stop codon, No significant transcription i nitiation inside the cap sequence of AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-PV4 was observed.