NS-3 Protein of the Junonia coenia Densovirus Is Essential for Viral DNA Replication in an Ld 652 Cell Line and Spodoptera littoralis Larvae

The genome of Junonia coenia densovirus (JcDNV) shares with members of the genus Densovirus the property of possessing structural (VP) and nonstructural (NS) genes in opposite orientations. The three NS genes located in the 5' half on one strand encode three NS proteins assumed to be involved in viral DNA replication: NS-1 and NS-2, which are common to all DNVs, and a 28-kDa polypeptide, NS-3, with a unique sequence. Whereas the essential role played by JcDNV NS-1 in viral DNA replication has been clearly established (C. Ding, M. Urabe, M. Bergoin, and R. M. Kotin, J. Virol. 76:338-345, 2002), nothing is known of the biological function(s) of NS-3. To investigate this function, we designed constructs derived from pBRJ, a plasmid encompassing an infectious sequence of JcDNV DNA (M. Jourdan, F. X. Jousset, M. Gervais, S. Skory, M. Bergoin, and B. Dumas, Virology 179:403-409, 1990), with partial or complete deletion of NS-3 sequence or with the ATG initiation codon mutated by site-directed mutagenesis. Transfection of these constructs to sensitive Ld 652 cells or Spodoptera littoralis larvae prevented the accomplishment of a productive cycle. We clearly established that the blocking of the replicative cycle in the absence of NS-3 expression occurred at the level of viral DNA replication. Replication of viral DNA could be restored by cotransfecting Ld 652 cells with a plasmid expressing JcDNV-NS-3 protein in trans. Time course analysis showed that NS-3 is produced early (6 h posttransfection) in the replicative cycle, and its production parallels that of replicative-form viral DNA. Finally, we present evidence that NS-1 and NS-2 proteins are synthesized at apparently the same levels whether or not NS-3 is expressed.