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ank Available online 9 July 2013 Keywords Coronavirus 90 IBV on the combination of the two and together with other two sites Ser190 and Ser192 into an infectious IBV clone did not affect recovery of the recombinant viruses containing the mutations A mutant virus rIBV Nm4 carrying the four Ala substitutions grew at a similar if not better growth rate as wild type virus virus kb length set A mRN the middle domain is an RNA binding domain capable of binding common feature prime function RNA to form a Davies et al Riso et al 1996 replication of the transcription Almaz n et al Tahara et al might inhibit stimulate strong humoral and cellular immune response making it Contents lists available at ScienceDirect Virology Virology 444 2013 225 232 for phosphorylation This protein contains several basic amino 1 Equal contributors a potential vaccine candidate Kim et al 2004 Coronaviruse N protein is an extensively phosphorylated and highly basic protein It varies from 377 to 455 amino acids in length and has high serine content 7 11 as potential targets 0042 6822 see front matter Li et al 2005 Of this host cell proliferation or delay cell growth possibly by disrupting cytokinesis Chen et al 2002 Wurm et al 2001 It can also form of polyproteins 1a and 1ab which are processed by virus encoded proteinases into at least 15 putative nonstructural pro teins NSP1 NSP16 Fang et al 2008 2010 The four structural proteins spike S envelope E membrane M and nucleocapsid N are encoded by subgenomic mRNAs In addition several putative nonstructural proteins such as 3a 3b 6 7a 7b 8a 8b 9b are also encoded by subgenomic mRNAs Snijder et al 2003 Thiel et al 2003 Coronavirus N protein contains multiple functional domains Sequence comparisons and structural studies have identi ed three nucleolar localization has been shown to be a of the coronavirus family Wurm et al 2001 The of the protein is to associate with the genomic ribonucleoprotein complex RNP and viral core 1981 Escors et al 2001 Narayana et al 2000 The protein may also play an important role in the the genomic RNA Chang and Brian 1996 and in and translation of subgenomic RNAs sgRNA 2004 Baric et al 1988 Stohlman et al 1988 1994 Z iga et al 2010 In addition N protein nomic mRNA species mRNA2 9 is expressed The genome length mRNA1 encodes two overlapping replicase proteins in the expressing the N protein it localizes either to the cytoplasm alone or to the cytoplasm and nucleolus Hiscox et al 2001 This Phosphorylation ATR Chk1 pathway Replication Introduction Coronavirus is an enveloped positive sense RNA genome of 27 30 with coronavirus a 3 coterminal nested including the genome length mRN This study reveals a cellular kinase responsible for phosphorylation of a coronavirus N protein at two positions and the functional consequence of this modi cation on coronavirus replication Tan et al 2012 Zhou et al 1996 The motifs for ribosome binding and nucleolar localization signals have been assigned to domain III Wurm et al 2001 More recently Chang et al 2009 proposed that all coronavirus N proteins may share a similar modular organization In cells N protein Identi cation of two ATR dependent phosphory on coronavirus nucleocapsid protein with in viral replication and infectivity in cultur Shouguo Fang a b 1 Linghui Xu b 1 Mei Huang b 1 Fr a Agricultural School Yangtze University 266 Jingmilu Jingzhou City Hubei Province 434025 b School of Biological Sciences Nanyang Technological University 60 Nanyang Drive Singapore article info Article history Received 12 April 2013 Returned to author for revisions 27 April 2013 Accepted 10 June 2013 abstract Coronavirus encodes an extensi studies have identi ed Ser1 infectious bronchitis virus Ser379 sites is dependent activated during IBV replication journal homepage lation sites nonessential functions ed cells Qisheng Li b D X Liu b n China 637551 Singapore vely phosphorylated and highly basic nucleocapsid N protein Previous Ser192 Thr378 and Ser379 as the phosphorylation sites for coronavirus N protein In this study we show that phosphorylation at Thr378 and ataxia telangiectasia mutated ATM and Rad3 related ATR a kinase Introduction of Ala substitutions at these two sites individually in yviro may play a role in the self association and homo oligomerization studied ATR substrate on Ser317 and Ser345 and replication protein A2 RPA2 a component of the heterotrimeric RPA com plex on Ser4 8 in IBV infected cells Xu et al 2011 The speci c residues for the ATM kinase activity including Chk2 on Thr68 and ATM on Ser1981 however were not detected in the same infected cells demonstrating the activation of the ATR Chk1 pathway during IBV infection Xu et al 2011 During this study we noted that when cells were infected with IBV at high multiplicity of infection and an excess amount of samples was loaded a batch of pChk1 speci c antibodies was also able to detect clearly a band migrating slightly more rapidly than pChk1 in IBV infected cells but not in cells infected with the ultraviolet UV inactivated IBV at 8 and 12 h post infection Fig 1 It should be pointed out that this observation was not consistently made when pChk1 speci c antibodies from different sources were used It was also noted that apparently more pChk1 was detected in cells treated with UV inactivated IBV Fig 1 This increased detection of pChk1 may represent cellular contamination from the inoculums as the virus stocks used for UV irradiation were prepared by freezing thawing the infected cells and were not puri ed Re probing of the same membrane with antibodies against IBV S Fang et al Virology 444 2013 225 232226 of the N protein and its interference of host cell division Li et al 2005 Infectious Bronchitis Virus IBV a prototype coronavirus is the etiological agent of infectious bronchitis which impairs the respiratory and urogenital tracts of chickens Cavanagh 2007 Similar to other coronaviruses IBV N protein is heavily phosphory lated By using mass spectroscopic analysis two conserved amino acid clusters Ser190Ser192 and Thr378Ser379 in IBV N protein were identi ed as the two regions for phosphorylation Chen et al 2005 Subsequently a total of six residues Ser162 Ser170 Thr177 Ser389 Ser424 and Thr428 were identi ed as phosphor ylation sites for mouse hepatitis virus MHV A59 N protein White et al 2007 and four phosphoserines at positions 9 156 254 and 256 were identi ed in transmissible gastroenteritis virus TGEV N protein Calvo et al 2005 In addition the host cell kinase s responsible for phosphorylation of coronavirus N protein is beginning to emerge One example is glycogen synthase kinase 3 which was shown to be involved in regulation of the phosphor ylation of severe acute respiratory syndrome coronavirus SARS CoV N protein Wu et al 2009 Current evidence suggests that phosphorylation may play important roles in regulation of the functions of coronavirus N protein These included regulation of its subcellular location viral RNA transcription particle assembly immunoreactivity and speci city Calvo et al 2005 Jayaram et al 2005 Shin et al 2007 Surjit et al 2005 Spencer et al 2008 However the functional relevance of N protein phosphor ylation has not been rigorously tested in the context of virus infected cells using an infectious clone system and host cell kinases responsible for phosphorylation of other coronavirus N proteins are not reported IBV infection perturbs cell cycle progression and arrests cell at the S and G2 M phases Li et al 2007 Wilson and Rangasamy 2001 Wurm et al 2001 More recently IBV infection was shown to induce DNA damage response and activation of the ataxia telangiectasia mutated ATM and Rad3 related ATR kinase checkpoint kinase 1 Chk1 pathway partly through interaction between coronavirus nsp13 and DNA polymerase delta Pol Xu et al 2011 In this study we show that phosphorylation at the Thr378 and Ser379 sites is dependent on ATR a kinase activated during IBV replication Introduction of Ala substitutions at these two sites individually in combination of the two and together with the other two sites Ser190 and Ser192 into an infectious IBV clone did not affect the recovery of recombinant viruses containing the mutations A mutant virus rIBV Nm4 carrying Ala substitutions at all the four previously identi ed sites grew at a similar if not better growth rate as wild type virus This study reveals a cellular kinase responsible for phosphorylation of a coronavirus N protein at two sites and the functional consequence of this modi cation on coronavirus replication in virus infected cells Results Speci c recognition of IBV N protein by phosphorylated Chk1 antibodies in IBV infected cells In a previous study we showed the phosphorylation of a acid rich regions functioning as nucleolar localization signals and RNA binding motifs and a serine arginine S R rich motif Li et al 2005 Fan et al 2005 Tan et al 2006 Biochemical characteriza tion and mutagenesis studies demonstrated that coronavirus N protein was also posttranslationally modi ed by covalent attach ment to the small ubiquitin like modi er SUMO Sumoylation number of ATR speci c substrates including Chk1 the best N protein detected the N protein at the exact position Fig 1 This result suggests that the protein band detected by anti pChk1 may be a phosphorylated form of the IBV N protein ATR dependent phosphorylation of IBV N protein A series of experiments was then carried out to con rm if this indeed represents a phosphorylated form of IBV N protein and if ATR is responsible for its phosphorylation First cells infected with IBV were analyzed by Western blot with antibodies against IBV N protein and ATM ATR substrates As shown in Fig 2a the full length 48 kDa IBV N protein and several smaller bands represent ing either premature or cleavage products derived from N protein Li et al 2005 were detected by Western blot with anti IBV N antibodies The 48 kDa band was also detected ef ciently by the anti ATM ATR substrate antibodies Fig 2a In addition a back ground band with unknown identity was also detected from both mock and IBV infected cells by this antibody Fig 2a The involvement of ATR in the phosphorylation of IBV N protein was then analyzed by addition of 10 and 15 M of Schisandrin B SchB a speci c ATR inhibitor functioning in IBV infected cells as demonstrated in a previous study Xu et al 2011 to IBV infected mock IBV IBV UV IBV UV Chk1 mock IBV IBV UV mock IBV N pChk1 4 h 8 h 12 h pChk1 S317 IBV N Chk1 actin 50 50 50 pN Fig 1 Detection of phosphorylated IBV N protein by Western blot analysis with anti phosphor Chk1 S317 antibodies H1299 cells were infected with live IBV IBV or UV inactivated IBV IBV UV at a multiplicity of infection of approximately 2 The mock treated cells were also included as a control At 4 8 and 12 h post infection cells were harvested and total lysates were subjected to immunoblotting assay The levels of phosphor Chk1 pChk1 S317 total Chk1 actin and IBV N were determined with appropriate antibodies Actin serves as a loading control and IBV N protein as a marker of IBV infection S Fang et al Virology 444 2013 225 232 227 cells at 4 h post infection To more speci cally analyze the full length IBV N with an unrelated antibody a recombinant IBV rIBV fN with a Flag tag added to the N terminus of the IBV N protein was constructed and used in this study The recombinant virus showed very similar growth kinetics as wild type IBV Fig 2b The cells were harvested at 24 h post infection and analyzed by Western blot As observed in our previous study Xu et al 2011 SchB exhibited a certain level of inhibitory effect on IBV replication IBV N protein was reduced to 78 and 55 when 10 and 15 M respectively of SchB were added as revealed by Western blot using anti Flag antibodies and densitometry analyses Fig 2c Probing of the same membrane with antibodies against ATM ATR substrates detected much less phosphorylated N protein in the presence of SchB Fig 2c Quanti cation of the band intensities by densitometry after normalizing to the total N protein showed that the ATR dependent phosphorylation of N protein was reduced to 47 and 2 after addition of 10 and 15 MofSchB respectively Fig 2c The phosphorylation of IBV N protein by ATR was then analyzed in cells overexpressing the N protein Cells transfected with IBV N construct were subjected to UV irradiation at 22 h post transfection in the presence or absence of 10 M of SchB Western blot analysis with antibodies against IBN N and ATM ATR sub strates showed that signi cantly less phosphorylated IBV N pro tein was detected in the UV irradiated transfected cells in the presence of 10 M of SchB Fig 2d In the absence of 10 Mof SchB a 2 8 fold increase of the ATR dependent phosphoryla tion of N protein was detected in the transfected cells after Fig 2 Phosphorylation of IBV N protein by the activated ATR a Detection of phosphorylat antibodies H1299 cells were infected with IBV wt at a multiplicity of infection of appro infection cells were harvested and total lysates were subjected to immunoblotting assay antibodies b Analysis of the growth kinetics of wild type and rIBV fN viruses Monolay 32 h post inoculation Viral stocks were prepared by freezing thawing of the cells three cells on 96 well plates in triplicate with 10 fold serial dilution of each viral stock c Inhibition cells H1299 cells were infected with rIBV fN at a multiplicity of infection of approximatel Cells were harvested at 24 h post infection and total lysates were subjected to immunoblotting anti Flag and anti ATR ATR substrates antibodies respectively Tubulin was included as a by SchB in cells over expressing the protein H1299 cells were transfected with IBN N At subjected to UV irradiation Cells were harvested at 24 h post infection and total lysates N were determined with anti IBV N and anti ATR ATR substrates antibodies respectivel UV irradiation compared to that in the cells without UV irradiation Fig 2d In the presence of 10 M of SchB a 1 5 fold increase of the ATR dependent phosphorylation of N protein was detected by the same comparison Fig 2d These results would lend more support totheconclusionthatATRisoneof the kinases responsible for phosphorylation of IBV N protein Construction and recovery of recombinant IBV carrying Ala substitutions at six predicted and previously identi ed phosphorylation sites on IBV N protein To systematically map the ATR dependent phosphorylation of IBV N protein Ala substitutions at the four previously identi ed sites Ser190 Ser192 Thr378 and Ser379 as well as two potential new sites Ser65 and Thr109 predicted to be ATR substrates Fig 3a were carried out The Ala substitutions were then introduced into an IBV infectious clone either individually or in combination of two four ve or six and nine recombinant IBVs were recovered from these constructs Fig 3a All the nine mutant viruses were passaged in cells up to eight passages and their genetic stability was determined by nucleotide sequencing of the entire N protein coding region The sequencing results con rmed that the Ala substitutions were stably maintained in all the mutant viruses and no additional mutations were introduced into the N protein during passaging The growth properties of the recombinant viruses passage 8 were then deter mined As shown in Fig 3b very similar growth kinetics was found between wild type IBV and the mutant viruses One notable ed IBV N protein by Western blot analysis with anti ATR ATM substrate ximately 1 The mock treated cells were also included as a control At 24 hours post The levels of phosphorylated and total IBV N were determined with appropriate ers of H1299 cells were infected with the viruses and harvested at 0 4 8 16 24 and times and TCID50 of each viral stock was determined by infecting ve wells of Vero of ATR dependent phosphorylation of IBV N protein by SchB in IBV infected y 1 At 4 h post infection 10 and 15 M of SchB were added to the cultured media assay The levels of phosphorylated and total IBV N were determined with loading control d Inhibition of ATR dependent phosphorylation of IBV N protein 22 h post transfection 10 M of SchB was added to the culture media and cells were were subjected to immunoblotting assay The levels of phosphorylated and total IBV y Tubulin was included as a loading control difference was that all mutant viruses reached their peak titers at 32 h post infection while wild type virus reached the peak titer at 24 h post infection Fig 3b Mapping of the ATR dependent phosphorylation sites on IBV N protein by Ala substitutions Wild type and seven mutant viruses Nm1 7 passage 3 were rst used to infect H1299 cells Total cell lysates were prepared and analyzed by Western blot with antibodies against ATM ATR substrates The results showed that the ATR dependent phosphor ylation of N protein was detected only in cells infected with wild type Nm1 and Nm2 mutant viruses Fig 4a The same band was not detected in cells infected with other ve mutant viruses Nm3 Nm7 Fig 4a As all these ve mutant viruses contain Ala substitutions at Thr378 and Ser379 positions it suggests that phosphorylation of IBV N protein at these two sites is ATR dependent Re probing of the same membrane with pChk1 speci c antibodies detected a much reduced amount of the phosphorylated N protein in cells infected with Nm4 mutant virus and moderately reduced amounts of the phosphorylated N protein in cells infected with Nm3 Nm5 Nm6 and Nm7 mutant viruses respectively Fig 4a The additional two mutant viruses Nm8 and Nm9 containing the Ala substitutions at Thr378 and Ser379 respectively Fig 3a were then used to assess the relative contribution of the two residues to the ATR dependent phosphorylation of IBV N protein H1299 cells were infected with wild type Nm3 Nm8 and Nm9 mutant viruses and total cell lysates were prepared Western blot analysis of total cell lysates with antibodies against ATM ATR substrates showed once again antibodies under the conditions shown in Fig 4a and b The reason Fig 3 Construction and recovery of mutant IBV carrying Ala substitutions at the predicted and previously identi ed phosphorylation sites on IBV N protein a Diagram showing the positions of the four previously identi ed phosphoryla tion sites and two additional putative sites for ATR on IBV N protein The Ala substitutions in each mutant construct are shown b Analysis of the growth kinetics of wild type and the nine mutant viruses Monolayers of H1299 cells were infected with the viruses and harvested at 0 4 12 24 and 32 h post infection Viral stocks were prepared by freezing thawing of the cells three times and TCID50 of each viral stock was determined by infecting ve wells of Vero cells on 96 well plates with 10 fold serial dilution of each viral stock a wer H1 total S Fang et al Virology 444 2013 225 232228 Fig 4 Mapping of the ATR dependent phosphorylation sites on IBV N protein protein H1299 cells were infected with wild type and seven mutant IBV Nm1 Nm7 included as a control At 24 h post infection cells were harvested and total lysates and total IBV N were determined with anti IBV N ATM ATR substrates and pChk1 speci effects of Ala substitutions on the ATR dependent phosphorylation of IBV N protein multiplicity of infection of approximately 1 The mock treated cells were also included subjected to immunoblotting assay The levels of ATR dependent phosphorylated and respectively Actin was included as a loading control may be due to the relatively low abundance of pChk1 compared to the phosphorylated N protein in virus infected cells Characterization of IBVNm4 mutant virus carrying Ala substitutions at all the four previously identi ed phosphorylation sites on IBV N protein The growth properties of IBVNm4 mutant virus were then characterized For this purpose Vero cells in duplicate were infected with wild type and IBVNm4 at a multiplicity of infection of approximately 1 At 24 h post infection one set of cells was stained The effe