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To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-\u0394E attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4<jats:sup>+<\/jats:sup>and CD8<jats:sup>+<\/jats:sup>T cell counts in the lungs of BALB\/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates.<\/jats:p><jats:p><jats:bold>IMPORTANCE<\/jats:bold>Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaviruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated<jats:italic>in vivo<\/jats:italic>. Here, we show that small deletions and modifications within the E protein led to virus attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels, and enhanced CD4<jats:sup>+<\/jats:sup>and CD8<jats:sup>+<\/jats:sup>T cell counts<jats:italic>in vivo<\/jats:italic>, suggesting that these phenomena contribute to virus attenuation. The attenuated mutants fully protected mice from challenge with virulent virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development.<\/jats:p>","DOI":"10.1128\/jvi.03566-14","type":"journal-article","created":{"date-parts":[[2015,1,22]],"date-time":"2015-01-22T05:59:19Z","timestamp":1421906359000},"page":"3870-3887","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":120,"title":["Severe Acute Respiratory Syndrome Coronaviruses with Mutations in the E Protein Are Attenuated and Promising Vaccine Candidates"],"prefix":"10.1128","volume":"89","author":[{"given":"Jose A.","family":"Regla-Nava","sequence":"first","affiliation":[{"name":"Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog\u00eda (CNB-CSIC), Darwin 3, Campus Universidad Aut\u00f3noma de Madrid, Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose L.","family":"Nieto-Torres","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog\u00eda (CNB-CSIC), Darwin 3, Campus Universidad Aut\u00f3noma de Madrid, Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose M.","family":"Jimenez-Guarde\u00f1o","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog\u00eda (CNB-CSIC), Darwin 3, Campus Universidad Aut\u00f3noma de Madrid, Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raul","family":"Fernandez-Delgado","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog\u00eda (CNB-CSIC), Darwin 3, Campus Universidad Aut\u00f3noma de Madrid, Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Craig","family":"Fett","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Iowa, Iowa City, Iowa, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Casta\u00f1o-Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog\u00eda (CNB-CSIC), Darwin 3, Campus Universidad Aut\u00f3noma de Madrid, Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stanley","family":"Perlman","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Iowa, Iowa City, Iowa, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis","family":"Enjuanes","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog\u00eda (CNB-CSIC), Darwin 3, Campus Universidad Aut\u00f3noma de Madrid, Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta L.","family":"DeDiego","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog\u00eda (CNB-CSIC), Darwin 3, Campus Universidad Aut\u00f3noma de Madrid, Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2147"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa030747"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1085952"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(03)13967-0"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1085953"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1211721"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01244-13"},{"key":"e_1_3_2_9_2","volume-title":"Middle East respiratory syndrome coronavirus (MERS-CoV)\u2014update","author":"WHO","year":"2013","unstructured":"WHO. 2013. 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