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1、Virus Research 153 (2010) 299304 Contents lists available at ScienceDirect Virus Research journal homepage: Short communication SARS-coronavirus protein 6 conformations required to impede protein import into the nucleus Snawar Hussain, Tom Gallagher Department of Microbiology and Immunology, Loyola
2、University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA a r t i c l ei n f o Article history: Received 8 June 2010 Received in revised form 16 August 2010 Accepted 18 August 2010 Available online 26 August 2010 Keywords: SARS-CoV Protein 6 Nuclear import Classical NLS a b s t r a
3、c t The severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes eight accessory pro- teins. Accessory protein 6 is a 63-residue amphipathic peptide that accelerates coronavirus infection kinetics in cell culture and in mice. Protein 6 is minimally bifunctional, with an N-terminal lip
4、ophilic part implicatedinacceleratingviralgrowthandaC-terminalhydrophilicpartinterferingwithgeneralprotein import into the nucleus. This interference with nuclear import requires interaction between protein 6 and cellular karyopherins, a process that typically involves nuclear localization signal (N
5、LS) motifs. Here we dissected protein 6 using site-directed mutagenesis and found no evidence for a classical NLS. Fur- thermore, we found that the C-terminal tail of protein 6 impeded nuclear import only in the context of a lipophilic N-terminus, which could be derived from membrane proteins unrela
6、ted to protein 6. These fi ndings are discussed in the context of the proposed protein 6 structure. 2010 Elsevier B.V. All rights reserved. The coronaviruses (CoVs) are enveloped, plus-strand RNA virusesthatarewidespreadinnature,infectingbirdsaswellasland, sea and fl ying mammals, causing humans gas
7、trointestinal and res- piratorydiseasesofvaryingseverity.The30kbCoVRNAgenomes are all distinguished by conserved gene organizations. The CoVs encode well-described proteins required for virus entry, replica- tion and assembly, and also encode more enigmatic small proteins of unknown function that ar
8、e specifi c to one of the three CoV anti- genic groups (Stadler et al., 2003). Reverse genetic engineering methods have been used to ablate one or more of these smaller group-specifi c genes, and in general, these engineered manipu- lations have had little effect on in vitro CoV viabilities (Yount e
9、t al., 2005). These fundamental observations make it clear that CoVs encode “accessory” proteins that are unnecessary for virus amplifi - cationincellculturebutarepresumablyoperatinginmorecomplex in vivo environments to maintain these viruses in nature. Exper- iments to discern CoV accessory protein
10、 structure and function may contribute generally to CoV virulence and zoonotic potential, and their genetically engineered modifi cation may be required to construct useful CoV vectors and attenuated CoV vaccines. SARS-CoV is the prototypic human pathogenic CoV. SARS-CoV is also the most complex kno
11、wn CoV with respect to accessory proteins, with eight accessory genes interspersed between those encodingvirionstructuralcomponents(Hussainetal.,2005;Rotaet al., 2003; Snijder et al., 2003). These accessory genes are present in Corresponding author. Tel.: +1 708 216 4850; fax: +1 708 216 9574. E-mai
12、l address: tgallaglumc.edu (T. Gallagher). SARS-CoVsisolatedfrombats,civetcats,raccoondogs,andhumans, suggesting important accessory functions in a variety of host envi- ronments (Li et al., 2005; Wang et al., 2005). There is clear evidence that these accessory genes are expressed (Chen et al., 2007
13、; Keng et al., 2006; Lu et al., 2006; Nelson et al., 2005; Pewe et al., 2005; Schaecher et al., 2007a,b; Tan et al., 2004; Yuan et al., 2006, 2005) but their functions remain somewhat obscure. Currently there are a range of suggested and indicated activities for accessory proteins, including viral s
14、tructural elements, ion channels, cell death induc- ers and interferon antagonists (Chen et al., 2007; Frieman et al., 2007; Hussain et al., 2008; Kopecky-Bromberg et al., 2007; Lu et al., 2006; Schaecher et al., 2007a,b; Tan et al., 2004; Yuan et al., 2005). Of all the CoV accessory proteins curren
15、tly under investiga- tion, the SARS-CoV protein 6 is arguably the best understood with respect to its structure and function. This intriguing 63 amino acid peptide is amphipathic in its amino-terminal 44 residues and very polar in the 20 residues comprising its C-terminus. Protein 6 is found entirel
16、y on cytoplasmic membranes (Geng et al., 2005), localizing on ER and Golgi organelles. Its membrane topology is N-endo C-endo, with a membrane-embedded stretch that is likely 6nm of alpha-helical structure (Netland et al., 2007; Zhou et al., 2010). With respect to function, protein 6 is a virulence factor, converting a sub-lethal infection of heterologous murine coron- avirus into a lethal infection and conferring growth advantages to murine coronavirus and also to SARS-CoV in infected host m
