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Minimum Determinants of Transmissible Gastroenteritis Virus Enteric Tropism Are Located in the N-Terminus of Spike Protein

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Minimum Determinants of Transmissible Gastroenteritis Virus Enteric Tropism Are Located in the N-Terminus of Spike Protein pathogens Article Minimum Determinants of Transmissible Gastroenteritis Virus Enteric Tropism Are Located in the N-Terminus of Spike Protein Carlos M. Sanchez y, Alejandro Pascual-Iglesias y, Isabel Sola , Sonia Zuñiga * and Luis Enjuanes * Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autónoma de Madrid, Darwin 3, 28049 Madrid, Spain; [email protected] (C.M.S.); [email protected] (A.P.-I.); [email protected] (I.S.) * Correspondence: [email protected] (S.Z.); [email protected] (L.E.); Tel.: +34-91-585-4526 (S.Z.); +34-91-585-4555 (L.E.) Both authors equally contributed to this work. y Received: 26 November 2019; Accepted: 14 December 2019; Published: 18 December 2019 Abstract: Transmissible gastroenteritis virus (TGEV) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide, that possesses both enteric and respiratory tropism. The ability to replicate in the enteric tract directly correlates with virulence, as TGEVs with an exclusive respiratory tropism are attenuated. The tissue tropism is determined by spike (S) protein, although the molecular bases for enteric tropism remain to be fully characterized. Both pAPN and sialic acid binding domains (aa 506–655 and 145–155, respectively) are necessary but not sufficient for enteric tract infection. Using a TGEV infectious cDNA and enteric (TGEV-SC11) or respiratory (TGEV-SPTV) isolates, encoding a full-length S protein, a set of chimeric recombinant viruses, with a sequential modification in S protein amino terminus, was engineered. In vivo tropism, either enteric, respiratory or both, was studied by inoculating three-day-old piglets and analyzing viral titers in lung and gut. The data indicated that U655>G change in S gene (S219A in S protein) was required to confer enteric tropism to a respiratory virus that already contains the pAPN and sialic acid binding domains in its S protein. Moreover, an engineered virus containing U655>G and a 6 nt insertion at position 1124 (Y374-T375insND in S protein) was genetically stable after passage in cell cultures, and increased virus titers in gut by 1000-fold. We postulated that the effect of these residues in enteric tropism may be mediated by the modification of both glycosaminoglycan binding and S protein structure. Keywords: transmissible gastroenteritis virus; tropism; recombinant virus; enteric tropism 1. Introduction Porcine enteric coronaviruses (CoVs) are one of the main threats for porcine industry worldwide, as acute infectious diarrhea is a major cause of high morbidity and mortality in piglets [1]. Transmissible gastroenteritis virus (TGEV) is one of the five enteric porcine CoVs described so far [2]. TGEV is classified in subgenus Tegacovirus of genus Alphacoronavirus, included in the family Coronaviridae into the order Nidovirales [3,4]. CoVs contain the largest known genome among RNA viruses, consisting in a single-stranded, positive-sense, 50-capped and polyadenylated RNA molecule of around 28 kb in length [5]. TGEV genomic RNA (gRNA) encodes replicase polyproteins (pp1a and pp1ab), and a set of structural and accessory genes in the order 50-S-3a-3b-E-M-N-7-30 [6]. TGEV has both enteric and respiratory tropism, in contrast to other porcine enteric CoVs, making this virus a good model to analyze the molecular determinants of coronavirus tissue tropism. Pathogens 2020, 9, 2; doi:10.3390/pathogens9010002 www.mdpi.com/journal/pathogens Pathogens 2020, 9, x FOR PEER REVIEW 2 of 13 Pathogens 2020, 9, 2 2 of 13 both enteric and respiratory tropism, in contrast to other porcine enteric CoVs, making this virus a good model to analyze the molecular determinants of coronavirus tissue tropism. SpikeSpike (S)(S) proteinprotein isis thethe mayormayor structuralstructural proteinprotein inin CoVCoV envelopeenvelope andand isis involvedinvolved inin receptorreceptor bindingbinding andand membranemembrane fusionfusion [[77––1010].]. In addition,addition, S proteinprotein isis thethe mainmain inducerinducer ofof neutralizingneutralizing antibodiesantibodies and,and, indeed,indeed, manymany CoVCoV vaccinesvaccines areare basedbased inin thethe expressionexpression ofof SS proteinprotein oror SS proteinprotein domainsdomains [[1111––1414].]. We andand othersothers havehave demonstrateddemonstrated that,that, byby modifyingmodifying SS proteinprotein sequence,sequence, CoVCoV tropismtropism isis alteredaltered [[1515––1818].]. Moreover,Moreover, speciesspecies tropismtropism couldcould alsoalso bebe changedchanged byby introducingintroducing SS proteinprotein sequencessequences inin thethe genomesgenomes ofof distantlydistantly relatedrelated CoVsCoVs [[1919––2222].]. PreviousPrevious workwork fromfrom ourour group,group, usingusing targetedtargeted recombinationrecombination betweenbetween aa respiratoryrespiratory helperhelper virusvirus (TGEV-PTV)(TGEV-PTV) andand aa minigenomeminigenome derivedderived fromfrom anan entericenteric TGEV,TGEV,showed showed thatthat recombinantrecombinant virusesviruses containingcontaining mutationsmutations inin thethe SS genegene regionregion betweenbetween ntnt 217217 andand 665665 lacklack entericenteric tropismtropism inin vivovivo[ 16[16]].. Moreover,Moreover, thethe analysisanalysis ofof recombinantrecombinant virusesviruses betweenbetween enteric enteric TGEV-PUR46-MAD TGEV-PUR46-MAD and respiratoryand respiratory TGEV-PTV, TGEV determined-PTV, determined that nucleotides that atnucleotides positions 214at positions and 655 of214 S geneand 655 were of determinantsS gene were determinants of the enteric of tropism the enteric [15]. tropism [15]. PorcinePorcine respiratoryrespiratory coronaviruscoronavirus (PRCV)(PRCV) isis aa TGEVTGEV mutantmutant thatthat naturallynaturally appearedappeared in thethe field,field, lackinglacking enteric enteric tropism tropism [23 [23,24],24]. PRCV. PRCV contains contains a 227 a amino227 amino acid deletionacid deletion in the N-terminusin the N-terminus of S protein of S (Figureprotein1 ),(Figure strongly 1), suggestingstrongly suggesting that this regionthat this of region TGEV Sof protein TGEV S is protein important is important for the enteric for the tropism. enteric Intropism. addition, In addition, we have previouslywe have previously shown that shown S protein that S binding protein tobinding the host to cellthe host receptor, cell receptor, the porcine the aminopeptidaseporcine aminopeptidase N (pAPN), N (pAPN) was not, was enough not enough to determine to determine the enteric the enteric tropism, tropism, as both as TGEVboth TGEV and PRCVand PRCV bind bind pAPN pAPN [10]. [10] By. comparing By comparing TGEV TGEV and and PRCV PRCV binding binding to cellto cell surface surface sialoglycoproteins, sialoglycoproteins, it wasit was proposed proposed that that TGEV TGEV binding binding to to this this type type of of molecules molecules may may facilitate facilitateenteric enteric tracttract infectioninfection [[25]25].. Nevertheless,Nevertheless, thethe sialicsialic acidacid bindingbinding domaindomain identifiedidentified inin TGEVTGEV SS proteinprotein isis conservedconserved inin severalseveral TGEV-derivedTGEV-derived respiratory respiratory viruses viruses with with a a full-lengthfull-lengthS S gene,gene, suchsuch asas TGEV-PTVTGEV-PTV (Figure(Figure1 1),), suggesting suggesting thethe existenceexistence of of additional additional determinants determinants of enteric of enteric tropism tropism in the in S protein.the S protein. PRCV genome PRCV alsogenome contains also deletionscontains deletions in the C-terminus in the C- ofterminus S gene, of in S genes gene, 3a, in 3b,genes and 3a, the 3b ORF1a, and the region ORF1a encoding region nsp3.encoding Then, nsp3. the contributionThen, the contribution of other S proteinof other domains S protein or domains other viral or other proteins viral to proteins enteric tropismto enteric was tropism for a long was timefor a anlong open time question an open [26 ].question In fact, the[26] relevance. In fact, ofthe S proteinrelevance N-terminus of S protein in TGEV N-terminus enteric tropismin TGEV has enteric even recentlytropism beenhas even questioned recently [27 been]. questioned [27]. Figure 1. Transmissible gastroenteritis virus (TGEV) spike (S) protein domains. The bar in the upper Figure 1. Transmissible gastroenteritis virus (TGEV) spike (S) protein domains. The bar in the upper part represents the S protein structure from a reference strain of an enteric TGEV (GenBank accession part represents the S protein structure from a reference strain of an enteric TGEV (GenBank accession number AJ271965). The numbers above each bar indicate amino acid positions. S1 and S2 domains are number AJ271965). The numbers above each bar indicate amino acid positions. S1 and S2 domains indicated. SP, signal peptide (red); HR, heptad-repeat motifs (purple); TM, transmembrane domain are indicated. SP, signal peptide (red); HR, heptad-repeat motifs (purple); TM, transmembrane (dark blue). The bars below represent a detail of N-terminus for enteric (C11) and respiratory [porcine domain (dark blue). The bars below represent a detail of N-terminus for enteric (C11) and respiratory respiratory coronavirus (PRCV), Purdue type virus (PTV)] TGEV-derived viruses, as previously defined [porcine respiratory coronavirus (PRCV), Purdue type virus (PTV)] TGEV-derived viruses, as in [16]. Several motifs are indicated: Antigenic sites C, B, D, and A (green) as previously defined inpreviously [9,28]; porcine defined aminopeptidase
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