Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of two copies of VP23 and one copy of VP19C. Here, we identified a non-classical NLS of VP19C between aa 50 and 61, and the nuclear import of VP19C was mediated by RanGTP and importin b1-, but not importin a5-, dependent pathway. Additionally, recombinant virus harbouring this NLS mutation (NLSm) replicates less efficiently as wild-type. These data strongly suggested that the nuclear import of VP19C is required for efficient HSV-1 production.Herpes simplex virus type 1 (HSV-1), a typical alphaherpesvirus, causes a spectrum of diseases, including herpes labialis, herpes keratitis and herpes encephalitis, which can be lethal . VP19C is a structural protein of the HSV-1 viral particle, which forms a triplex consisting of two copies of VP23 and one copy of VP19C (Chowdhury & Batterson, 1994;Kim et al., 2011;Solé et al., 2007;Spencer et al., 1998). There are 320 hetero-trimeric triplex molecules per capsid, which are assembled in the cytoplasm and transported to the nucleus, where higherorder capsid shells are assembled (Okoye et al., 2006;Saad, 2003;Solé et al., 2007;Trus et al., 1996). VP19C is essential for assembly of the capsid and if it is absent capsid shells do not form (Person & Desai, 1998). A previous report has described that the nuclear targeting property of VP19C is required for nuclear transport of VP23 (Rixon et al., 1996), which is incapable of localizing to the nucleus on its own. A previous study has demonstrated that the N terminus of VP19C was required for its nuclear import (Adamson et al., 2006); however, the precise NLS responsible for its nuclear targeting was still elusive.As previously reported (Rixon et al., 1996), VP19C exhibited a predominantly, if not exclusively, nuclear localization in HSV-1-infected cells, which was demonstrated by using a fixation procedure. However, it is well known that some fixation protocols may alter the subcellular localization of proteins, resulting in misleading conclusions. As an alternative method to investigate the intracellular distribution of VP19C, a living-cell fluorescence microscopy technique and enhanced-yellow-fluorescent protein (EYFP)-tagged variants were applied (Cai et al., 2011;Xing et al., 2010 Xing et al., , 2011b. To investigate the subcellular localization of VP19C in the absence of other viral proteins, pVP19C-EYFP was constructed and transfected into COS-7 cells as described previously (Li et al., 2011a). The fluorescence of VP19C-EYFP was predominantly restricted to the nucleus (Fig. 1c), which is consistent with the previous report (Rixon et al., 1996).It was previously reported that the first 56 aa of VP19C was important for its nuclear localization (Adamson et al., 2006); however, the precise NLS was still unknown. To map the amino acid sequence within VP19C responsible for its nuclear localization, a series of deletion mutants encompassing aa 1-30, 30-50, 41-52, 50-61 and 1-61 each fused to EYFP were constructed (Fig. 1a). Their expressi...