White spot syndrome virus (WSSV) is a fatal pathogen of shrimp and has a circular, double-stranded DNA genome of about 300 kb (18, 22). Unique aspects of its morphology and genome caused this virus to be classified in a new virus family, Nimaviridae (3, 11). The intact virion is comprised of at least 59 structural proteins, the localization of 44 of which in viral particle have been established; 35 are defined as envelope proteins (including tegument proteins) and nine as nucleocapsid proteins (1, 9, 10, 14, 15, 17, 20, 21).
Like most DNA viruses, WSSV replicates in the nucleus of host cells (13). However, due to the lack of permissive cell lines, the life cycle of WSSV is com-pletely unknown. After entering the cell and passing through the cytosol, entry of the viral genome into the nucleus is a prerequisite for viral genome expression and replication, and is thus essential for initiating the viral life cycle. The available evidence indicates that most nuclear replicating viruses enter the nucleus through nuclear pore complexes (NPCs) (8, 19). Tar-geting and transport of viral genomes to the nucleus depends on nuclear localization sequences (NLSs), exposed on the surface of the capsid particle. This process relies on assistance from cellular import receptors in the form of importins (karyopherins) (8, 19). NLSs are short stretches of residues that mediate the transport of nuclear proteins into the nucleus (2). There are two types of NLS: monopartite NLSs which comprise a short stretch of basic amino acids (7) and bipartite NLSs, consisting of two stretches of basic amino acids separated by a spacer of 10-12 amino acids (12).
A recent report proposed that VP664, VP51C, VP60B and VP15 were the major components of the nucleocapsid. VP664 and VP15 both appear in the nucleocapsid and VP15 is a core protein, involved in the packaging of the WSSV genome in the nucleo-capsid (15, 16, 20, 23). VP15 would therefore be expected to play an important role in transporting the viral genome to the nucleus. Three bipartite NLSs NLS1 (aa 11-27, RRGSKKRSTTAGRISKR), NLS2 (aa 33-49, KKRAGKKSSTVRRRSSK) and NLS3 (aa 44-60, RRRSSKSGKKSGARKSR) (the conserved basic residues are shown in bold letters) were detected in the sequence of VP15, using the computer program ScanProsite (4), but whether or not it is a nuclear transport protein remains unclear. In this study, we characterized the nuclear localization function of VP15 and its three functional bipartite NLSs.
Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus *
- Received Date: 05 December 2008
- Accepted Date: 12 December 2008
Abstract: The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.