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Contents lists available at ScienceDirect Virology journal homepage The OC43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and CNS pathology Jenny K Stodola 1 Guillaume Dubois 1 Alain Le Coupanec Marc Desforges Pierre J Talbot Laboratory of Neuroimmunovirology INRS Institut Armand Frappier Laval Qu bec Canada ARTICLE INFO Keywords HCoV OC43 Coronavirus E protein Transmembrane domain PDZ binding motif Virus production Pathogenesis ABSTRACT The OC43 strain of human coronavirus HCoV OC43 is an ubiquitous respiratory tract pathogen possessing neurotropic capacities Coronavirus structural envelope E protein possesses speci c motifs involved in protein protein interaction or in homo oligomeric ion channel formation which are known to play various roles in cluding in virion morphology assembly and in cell response to infection and or virulence Making use of re combinant viruses either devoid of the E protein or harboring mutations either in putative transmembrane domain or PDZ binding motif we demonstrated that a fully functional HCoV OC43 E protein is rst needed for optimal production of recombinant infectious viruses Furthermore HCoV OC43 infection of human epithelial and neuronal cell lines of mixed murine primary cultures from the central nervous system and of mouse central nervous system showed that the E protein is critical for e cient and optimal virus replication and propagation and thereby for neurovirulence 1 Introduction Coronaviruses are widespread RNA viruses of the Nidovirales order Coronaviridae family most often associated with human and veterinary respiratory infections de Groot et al 2012 Of the six human infecting coronavirus strains four HCoV 229E HCoV NL63 HCoV HKU1 and HCoV OC43 are currently co circulating and elicit respiratory illnesses Vabret et al 2009 Coronaviruses also represent a signi cant public health concern due to the recent zoonotically emerged highly patho genic species SARS corovonavirus SARS CoV Drosten et al 2003 Ksiazek et al 2003 in 2002 2003 and since 2012 Middle East re spiratory syndrome coronavirus MERS CoV Zaki et al 2012 loca lized to the Arabian Peninsula but with sporadic travel related out breaks worldwide In addition to their respiratory tropism human coronaviruses have been detected concurrently with severe and acute neurological symptoms Arabi et al 2015 Morfopoulou et al 2016 Yeh et al 2004 and shown to naturally infect the central nervous system CNS Arbour et al 2000 Gu et al 2005 Xu et al 2005 with neurons demonstrated as the main target of infection in HCoV OC43 Bonavia et al 1997 Favreau et al 2012 Jacomy et al 2006 Jacomy and Talbot 2003 and SARS CoV Gu et al 2005 Xu et al 2005 Coronaviruses represent the largest known enveloped RNA single stranded positive sense viruses with a genome of approximately 30 kb de Groot et al 2012 The viral envelope is composed of four or ve proteins the spike S membrane M envelope E and hemagglu tinin esterase protein HE the latter in some coronaviruses genus such as HCoV OC43 Coronavirus E proteins are 74 109 amino acids in length 84 amino acids for HCoV OC43 and share only a small amount of sequence identity between coronavirus species However its sec ondary structure composed of a short N terminal domain followed by a single hydrophobic transmembrane domain TMD and hydrophilic cytoplasmic tail remains overall conserved and is suggested to be more important than sequence for function Kuo et al 2007 Torres et al 2005 The importance of the presence of the E protein in the viral envelope is emphasized by the fact that there are only about twenty E molecules incorporated within the virion structure Godet et al 1992 Liu and Inglis 1991 Yu et al 1994 and deletion of the protein can either completely prevent the production of detectable infectious vir ions Almaz n et al 2013 Curtis et al 2002 Ortego et al 2007 2002 or signi cantly reduce infectious virus titers DeDiego et al 2008 2007 Kuo et al 2007 Kuo and Masters 2003 The majority of the coronavirus E protein in the infected cell is lo calized within the secretory pathway between the membranes of the endoplasmic reticulum ER Golgi and intermediate compartment https doi org 10 1016 j virol 2017 12 023 Received 15 November 2017 Received in revised form 8 December 2017 Accepted 20 December 2017 Correspondence to 531 Boulevard des Prairies Laval Qu bec Canada H7V 1B7 1 Contributed equally to this work E mail addresses marc desforges iaf inrs ca M Desforges pierre talbot iaf inrs ca P J Talbot Virology 515 2018 134 149 0042 6822 2017 Elsevier Inc All rights reserved T between them ERGIC Cohen et al 2011 Nieto Torres et al 2011 Venkatagopalan et al 2015 It is in this intracellular region that ad ditional functions mediated by various domains of the coronavirus E proteins are proposed to occur Homo pentameric oligomerization of the E protein TMD in membranes to form ion channels called vir oporins has been predicted for several coronaviruses Torres et al 2005 and extensively studied for species such as SARS CoV Nieto Torres et al 2014 Pervushin et al 2009 or avian infectious bronchitis virus IBV Ruch and Machamer 2012 Westerbeck and Machamer 2015 Another domain found at the extreme C terminal end of the E protein a PDZ domain binding motif PBM has also been predicted for several coronavirus species Jimenez Guarde o et al 2014 This protein protein interaction motif capable of interrupting normal cel lular functions has been demonstrated in other viruses to play im portant roles in replication dissemination in the host and pathogenesis Javier and Rice 2011 The multiple properties of coronavirus E pro teins have not yet been fully investigated or explained and can at times di er between coronavirus species The multifunctionality of the E protein could be explained by the presence of two distinct pools monomeric versus homo oligomeric states present in the infected cell Westerbeck and Machamer 2015 Furthermore the di erent motifs found within the protein Jimenez Guarde o et al 2015 could med iate di erent speci c functions The coronavirus E protein was also recently recognized as an important virulence factor for the SARS CoV Jimenez Guarde o et al 2014 where deletion of the whole or part of the protein led to an attenuated pathology in mouse lungs DeDiego et al 2008 2007 attenuation which was later linked to the E protein TMD and PBM Jimenez Guarde o et al 2014 Nieto Torres et al 2014 HCoV OC43 represents a circulating strain of human coronavirus causing respiratory illness which is naturally capable of invading the CNS where neurons are preferentially targeted for infection In this study we demonstrate that the fully functional HCoV OC43 E protein harboring speci c TMD and PBM is critical in infectious virus pro duction and dissemination in epithelial and neuronal cell cultures and in the murine CNS and that it is a determinant of neurovirulence a rst demonstration for this coronavirus species 2 Results 2 1 Deletion of HCoV OC43 E protein abrogates infectious virion production introduces a strong selection pressure for reversion in progeny production In order to evaluate the importance of the HCoV OC43 E protein in infectious virion production a stop codon was introduced at the be ginning of the E gene of our cDNA infectious clone pBAC OC43 FL St Jean et al 2006 preventing corresponding full length E protein pro duction in the resultant recombinant virus Fig 1A Transfection of BHK 21 cells with the pBAC OC43FL led to the detection of reference HCoV OC43 recombinant infectious virus rOC ATCC whereas trans fection with the pBAC OC43 E Stop mutant did not lead to any de tectable infectious virus rOC E Stop Fig 1B To con rm that the inability to detect infectious viral particles was due to the lack of E protein expression we wished to verify whether viral production could be rescued with wild type E protein Transfection of a plasmid containing the reference HCoV OC43 E gene pcDNA OC E in BHK 21 cells clearly showed via Western blot assay WB that the E protein was produced compared to an empty plasmid condition Fig 1C Subsequently a transient co transfection was con ducted in the same cells with pBAC E Stop and pcDNA OC E and by making use of a monoclonal antibody against the S protein of HCoV OC43 we con rmed that the co transfection did not a ect transfection e ciency and that the viral S protein was produced at equivalent levels in cells transfected with pBAC OC43 FL alone or pBAC E Stop with pcDNA OC E or empty plasmid Fig 1D Following the co transfection infectious particles production was rescued to detectable levels in a dose dependent manner Fig 1E Viral RNA was harvested and cDNA sequenced to con rm that the infectious particles detected after transfection corresponded to rOC ATCC and rOC E Stop mutant data not shown As we were able to rescue infectious particles production through transient complementation we wondered whether this resultant virus still lacking the E gene could be ampli ed further in subsequent pas sages To this end we ampli ed the viral stocks of all transfected plasmids three times without trans complementation on HRT 18 epi thelial cells each time normalizing to the lowest detectable viral titer to infect cells at an identical MOI for all recombinant viruses Fig 1F Throughout the ampli cation process we were consistently unable to detect infectious viral particles issuing from viral stocks of rOC E Stop Ampli cations of initially complemented viral stocks of rOC E Stop led to detectable titers which decreased over the course of each subsequent ampli cation compared to rOC ATCC Sequencing of viral RNA con rmed that the E gene in the viral rOC E Stop stocks contained the introduced stop codon at each ampli cation step data not shown These results demonstrate that production of progeny infectious HCoV OC43 virions is still possible in the absence E protein however the e ciency of the process is dramatically diminished Interestingly when conducting independent experiments following the same experimental approach the titers of initially complemented rOC E Stop sometimes increased substantially after two or three am pli cations approaching reference virus titer levels after three rounds of ampli cation on HRT 18 cells Fig 1F Sequence analysis of the E gene of the corresponding viral stocks revealed that a reversion of se quence appeared at the position where the stop codon had been initially introduced representing reversion to wild type or new amino acids Fig 1G Taken together these data demonstrate that the HCoV OC43 E protein is critical for e cient infectious virion production in epithe lial cells 2 2 Neuronal cells are susceptible to infection with HCoV OC43 lacking E protein but progeny virus production is severely inhibited HCoV OC43 is neuroinvasive Arbour et al 2000 and neurotropic with the neuron being the main target of infection in the CNS Jacomy et al 2006 Jacomy and Talbot 2003 Therefore we sought to in vestigate whether the absence of the E protein would modify these neurotropic capacities by infecting a susceptible di erentiated human neuronal cell line LA N 5 or mixed primary cultures of murine CNS cells Initially complemented rOC E Stop previously recovered from transfection on BHK 21 cells P0 was used for infection and infectious viral titers determined over a period of 72 h post infection hpi This revealed an important decrease of infectious virus production for human cells Fig 2A which was exacerbated in primary murine cells where virus titers were under the limit of detection Fig 2B However in these primary cultures low levels of infected cells were visualized by immuno uorescence IFA where we detect the viral S protein sug gesting that infection was possible even for the complemented rOC E Stop virus but that production of new infectious progeny and eventual propagation were severely inhibited compared to wild type virus Fig 2C 2 3 HCoV OC43 E protein putative transmembrane domain integrity is important for e cient infectious virion production and e cient infection of neuronal cells The transmembrane domain TMD of some coronavirus E protein is known to homo oligomerize in membranes and appears to modulate infectious virus production Nieto Torres et al 2014 In order to de termine the e ect of HCoV OC43 E protein TMD on virus production in cell culture pBAC OC43FL was modi ed at a key amino acid position previously identi ed in other coronaviruses to be critical for the J K Stodola et al Virology 515 2018 134 149 135 stability of this speci c domain Nieto Torres et al 2014 Ruch and Machamer 2012 and compared against wild type virus during infec tion of cells The large polar glutamine at position 17 of the HCoV OC43 E protein putative TMD was modi ed into a smaller non polar alanine pBAC E TM Q17A in order to diminish any possible ion channel selectivity conveyed by this amino acid Pervushin et al 2009 at the opening of the putative ion channel Transfection of transmembrane mutant in BHK 21 cells yielded detectable virus titers of rOC E TM Q17A which was further ampli ed on HRT 18 cells P1 to signi cantly lower titers compared to reference virus Fig 3A Infection of human LA N 5 cells and mixed primary cultures of mouse CNS cells showed a similar virus production kinetic Fig 1 The HCoV OC43 E protein is critical for infectious particle production A Representation of the full length HCoV OC43 genome found within the pBAC OC43 FL infectious clone top with a schematic representation of the HCoV OC43 E gene to be modi ed at various amino acid positions indicated at their relative positions within the protein bottom TM transmembrane PBM PDZ binding motif B Evaluation of infectious virus production corresponding to pBAC E Stop transfection of BHK 21 cells compared to pBAC OC43FL C Insertion of wild type E gene into pcDNA3 1 expression vector pcDNA OC E yielded corresponding HCoV OC43 E protein expression compared to empty vector D Visualization of transfection e ciency on BHK 21 cells of various conditions by immuno uorescence detection of HCoV OC43 S protein green nucleus staining with DAPI blue Subpanels a mock b pBAC OC43FL rOC ATCC c pBAC E Stop rOC E Stop 2 g pcDNA OC E d pBAC E Stop rOC E Stop 2 g pcDNA empty E Transient co transfection of pBAC E Stop and 1or 2 g pcDNA OC E in BHK 21 cells rescued detectable infectious virus in a dose dependent manner P 0 05 F Evaluation of infectious recombinant virus production after transient co transfection of pBAC E Stop and pcDNA OC E in BHK 21 cells BHK 0 The supernatants P0 were ampli ed three times by inoculation of HRT 18 cells HRT 1 3 Infectious viral titer di erences observed between experiments revealed by sequencing G the appearance of reversions at the position in the E gene where a stop codon was introduced are indicated by bold and underline LOD limit of detection cross indicates appearance of reversion s in the HCoV OC43 E gene in viral stocks as detected by sequencing J K Stodola et al Virology 515 2018 134 149 136 with an initial delay over the rst 24hpi in the cell free fraction Fig 3B and C left panels However in the cell associated fractions the amount of recovered infectious virus particles was almost identical to those of the reference virus Fig 3B and C right panels suggesting a possible defect in virus release In order to ascertain this potential de fect human LA N 5 cells were infected with either rOC ATCC or rOC E TM Q17A and both infectious titer and viral RNA copies associated to total viral particles in the cell free fraction were quanti ed at 16 h Fig 2 HCoV OC43 lacking E protein can infect neuronal cells but replication is severely impaired LA N 5 A and mixed primary mouse CNS cells B were infected with supernatant coming from BHK21 supernatant P0 and containing virus lacking E protein rOC E Stop pcDNA empty or initially complemented virus rOC E Stop pcDNA OC E Cell free and cell associated virus infectious titers were determined over 72 h post infection Representative of three di erent experiments Statistical signi cance was tested at 72hpi P 0 001 C IFA on infected mixed primary murine CNS cells over 48 h Green represents the microtubule associated protein 2 MAP2 staining in neurons red represents viral S protein J K Stodola et al Virology 515 2018 134 149 137 Fig 3 The putative HCoV OC43 transmembrane domain plays an important role in infectious virion production release replication and propagation in neuronal cells A Production of infectious virus after transfection of E protein transmembrane mutant in BHK 21 cells BHK 0 and ampli cation on HRT 18 cells HRT 1 B LA N 5 human neuronal cells and C mixed primary mouse CNS cells were infected with rOC E TM Q17A Cell free and cell associated virus fractions were recovered and tittered over 72 h The results show a representative experiment Statistical signi cance was tested at 72hpi P 0 01 P10 13 were the only ones to show signs of illness at 5 dpi To investigate the role of the E protein in the induction of HCoV OC43 induced neurological pathology 22 day old C57Bl 6 mice were intracerebrally infected with either rOC ATCC rOC E TM Q17A or rOC E PBM D82A V84A and the development of illness was monitored for 21 days after infection During this period only mice infected with the reference virus died all other mice survived the infection Fig 6B Moreover mice infected with rOC E PBM D82A V84A did not show any signi cant di erences of weight gain compared to sham infected mice Fig 6C nor did they show any sign of neurological disease compared to mice infected with the reference virus Fig 6D However mice in fected with rOC E TM Q17A showed an intermediate weight gain pro le between mice infected with the reference virus and the sham infected mice Fig 6C suggesting that these mice were developing a disease which was con rmed by the neurological symptoms developed by several mice although to a lesser extent than mice infected with the reference virus Fig 6D Mice infected with rOC E stop did not show any signs of illness whether in terms of weight gain or neurological symptoms Figure S3A C 2 7 HCoV OC43 E protein and its TMD and PBM are essential for e cient replication in the murine CNS As HCoV OC43 E protein and its functional domains modulate viral replication and propagation in human and murine neuronal cultures Figs 3 and 4 we examined if the di erences in neurovirulence ob served Fig 6 were also to be associated with defective infectious virus productions and propagation in the CNS Infection of 22 day old C57Bl 6 mice with reference and E protein mutants revealed that the J K Stodola et al Virology 515 2018 134 149 139 Fig 4 A functional PDZ domain binding motif at the C terminal end of the HCoV OC43 E protein is critical for e cient virus production and spread in neuronal cells A Production of infectious virus after transfection of various E protein mutants with fully or partially abrogated predicted PBM after transfection in BHK 21 cells BHK 0 and ampli cation on HRT 18 cells HRT 1 The results show a representative experiment Cell free and cell associated infectious virus production were determined at indicated timepoints after infection at an MOI of 0 05 on B LA N 5 cells and C mixed primary cultures of mouse CNS cells The results show a representative experiment Statistical signi cance was tested at 72hpi P 0 01 P 0 001 D The percentage of LA N 5 cells infected by the transmembrane mutant representative of viral propagation was quanti ed by immuno uorescence and cell pro ler software and compared to the reference virus over 72 h post infection LOD limit of detection J K Stodola et al Virology 515 2018 134 149 140 infectious titer in the brain Fig 7A and the spinal cord Fig 7B was signi cantly reduced for the TMD mutant This altered replication in the brain correlates with an extremely weak production in the spinal cord where infectious v