Short Communication Identification of a novel NLS of herpes simplex virus type 1 (HSV-1) VP19C and its nuclear localization is required for efficient production of HSV-1 You Li,3 Lei Zhao,3 Shuai Wang, Junji Xing and Chunfu Zheng Correspondence Chunfu Zheng zheng.alan@hotmail.com Received 8 March 2012 Accepted 23 May 2012 Molecular Virology and Viral Immunology Research Group, State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China 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 alphaher- pesvirus, causes a spectrum of diseases, including herpes labialis, herpes keratitis and herpes encephalitis, which can be lethal (Wang et al., 2012). VP19C is a structural protein of the HSV-1 viral particle, which forms a triplex consist- ing of two copies of VP23 and one copy of VP19C (Chowdhury & Batterson, 1994; Kim et al., 2011; Sole ´ 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 higher- order capsid shells are assembled (Okoye et al., 2006; Saad, 2003; Sole ´ 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 demon- strated 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 demon- strated 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 fluor- escence microscopy technique and enhanced-yellow-fluor- escent protein (EYFP)-tagged variants were applied (Cai et al., 2011; Xing et al., 2010, 2011b). To investigate the subcellular localization of VP19C in the absence of other viral proteins, pVP19C–EYFP was constructed and trans- fected into COS-7 cells as described previously (Li et al., 2011a). The fluorescence of VP19C–EYFP was predomi- nantly 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 expression was confirmed by Western blot analysis using anti-YFP antibody (Clontech) (Fig. 1b). Then the subcellular locali- zation of these fusion proteins was imaged in live cells. As shown in Fig. 1(c), subcellular distribution of VP19C aa 1– 61–EYFP was identical to that of VP19C–EYFP, with fluorescence enriched in the nucleus. While, aa 1–30– EYFP, aa 30–40–EYFP and aa 41–52–EYFP showed ident- ical pan-cellular localization to EYFP. Surprisingly, VP19C aa 50–61–EYFP distributed primarily to the nucleus (Fig. 1c), suggesting that aa 50–61 might be responsible for nuclear localization of VP19C. The function of a putative NLS can be tested by mutation (Li & Zhu, 2009). A protein containing a defective NLS fails to enter the nucleus. To validate whether the putative NLS was responsible for VP19C nuclear translocation, three arginines in the putative NLS were mutated to ala- nines (Fig. 1a). As a result, mutation of arginines signifi- cantly attenuated protein accumulation in the nucleus (Fig. 1c), suggesting that these arginines are important for nu- clear translocation of VP19C. Accordingly, we have identi- fied VP19C aa 50–61 (PRGSGPRRAAS) as a non-classical NLS that does not belong to any of the known classes of 3These authors contributed equally to this work. Journal of General Virology (2012), 93, 1869–1875 DOI 10.1099/vir.0.042697-0 042697 G 2012 SGM Printed in Great Britain 1869