The human SARS-CoV 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells

Virology
Volume 354, Issue 1, 10 October 2006, Pages 132-142

Choong-Tat Keng, Yook-Wah Choi, Matthijs R.A. Welkers, Daphne Z.L. Chan, Shuo Shen, Seng Gee Lim, Wanjin Hong, Yee-Joo Tan.

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesis.

Keywords

Severe acute respiratory syndrome (SARS), Coronavirus (CoV), Accessory proteins, Envelope (E) protein, 8a, 8b, 8ab

Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients

Clinical Immunology
Volume 120, Issue 2, August 2006, Pages 171-178

Li-Tao Yang, Hui Peng, Zhao-Ling Zhu, Gang Li, Zi-Tong Huang, Zhi-Xin Zhao, Richard A. Koup, Robert T. Bailer, Chang-You Wu

Abstract

The role of cell-mediated immunity in human SARS-CoV infection is still not well understood. In this study, we found that memory T-cell responses against the spike (S) protein were persistent for more than 1 year after SARS-CoV infection by detecting the production of IFN-γ using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4+ and CD8+ T cells were involved in cellular responses against SARS-CoV infection. Interestingly, most of SARS-CoV S-specific memory CD4+ T cells were central memory cells expressing CD45RO+ CCR7+ CD62L−. However, the majority of memory CD8+ T cells revealed effector memory phenotype expressing CD45RO− CCR7− CD62L−. Thus, our study provides the evidence that SARS-CoV infection in humans can induce cellular immune response that is persistent for a long period of time. These data may have an important implication in the possibility of designing effective vaccine against SARS-CoV infection, specifically in defining T-cell populations that are implicated in protective immunity.

Keywords

SARS, SARS-CoV, S protein, IFN-γ, Cellular immune response, Memory T cells

Accelerated induction of apoptosis in insect cells by baculovirus-expressed SARS-CoV membrane protein

FEBS Letters
Volume 580, Issue 16, 10 July 2006, Pages 3829-3834

Edited by Hans-Dieter Klenk

Chia-Wei Lai, Zun-Ren Chan, Ding-Gang Yang, Wen-Hsin Lo, Yiu-Kay Lai, Margaret Dah-Tsyr Chang, Yu-Chen Hu

Abstract

It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.

Keywords

Apoptosis, Baculovirus, Insect cell, Membrane protein, SARS-CoV

A recombinant baculovirus-expressed S glycoprotein vaccine elicits high titers of SARS-associated coronavirus (SARS-CoV) neutralizing antibodies in mice

Vaccine
Volume 24, Issue 17, 24 April 2006, Pages 3624-3631

Zhimin Zhou, Penny Post, Rick Chubet, Katherine Holtz, Clifton McPherson, Martin Petric, Manon Cox.

Abstract

A recombinant SARS-CoV spike (S) glycoprotein vaccine produced in insect cells in a pre-clinical development stage is described. A truncated version of S glycoprotein, containing only the ecto-domain, as well as a His-tagged full-length version were cloned and expressed in a serum-free insect cell line, ExpresSF+®. The proteins, purified to apparent homogeneity by liquid column chromatography, were formulated without adjuvant at 3, 9, 27, and 50 μg per dose in phosphate saline and used to immunize mice. Both antigens in each formulation elicited a strong immune response after two or three vaccinations with the antigen. Neutralizing antibody titers correlated closely with standard ELISA reactivity against the S glycoprotein. The truncated S protein was also formulated with an adjuvant, aluminum hydroxide, at 1 μg per dose (±adjuvant), and 5 μg per dose (±adjuvant). Significantly enhanced immune responses, manifested by higher titers of serum ELISA and viral neutralizing antibodies, were achieved in adjuvanted groups with fewer doses and lower concentration of S glycoprotein. These findings indicate that the ecto-domain of SARS-CoV S glycoprotein vaccine, with or without adjuvant, is immunogenic and induces high titers of virus neutralizing antibodies to levels similar to those achieved with the full S glycoprotein vaccine.

Keywords

SARS-CoV, Recombinant protein vaccine, Virus neutralization, Adjuvant, Aluminum hydroxide

Polymorphism of SARS-CoV Genomes

Acta Genetica Sinica
Volume 33, Issue 4, April 2006, Pages 354-364

SHANG Lei, QI, Yan, BAO, Qi-Yu, TIAN, Wei, XU, Jian-Cheng, FENG, Ming-Guang, YANG, Huan-Ming

Abstract

In this work, severe acute respiratory syndrome associated coronavirus (SARS-CoV) genome BJ202 (AY864806) was completely sequenced. The genome was directly accessed from the stool sample of a patient in Beijing. Comparative genomics methods were used to analyze the sequence variations of 116 SARS-CoV genomes (including BJ202) available in the NCBI Gen-Bank. With the genome sequence of GZ02 as the reference, there were 41 polymorphic sites identified in BJ202 and a total of 278 polymorphic sites present in at least two of the 116 genomes. The distribution of the polymorphic sites was biased over the whole genome. Nearly half of the variations (50.4%, 140/278) clustered in the one third of the whole genome at the 3′ end (19.0 kb-29.7 kb). Regions encoding Orf10–11, Orf3/4, E, M and S protein had the highest mutation rates. A total of 15 PCR products (about 6.0 kb of the genome) including 11 fragments containing 12 known polymorphic sites and 4 fragments without identified polymorphic sites were cloned and sequenced. Results showed that 3 unique polymorphic sites of BJ202 (positions 13 804, 15 031 and 20 792) along with 3 other polymorphic sites (26 428, 26 477 and 27 243) all contained 2 kinds of nucleotides. It is interesting to find that position 18379 which has not been identified to be polymorphic in any of the other 115 published SARS-CoV genomes is actually a polymorphic site. The nucleotide composition of this site is A (8) to G (6). Among 116 SARS-CoV genomes, 18 types of deletions and 2 insertions were identified. Most of them were related to a 300 bp region (27 700–28 000) which encodes parts of the putative ORF9 and ORF10–11. A phylogenetic tree illustrating the divergence of whole BJ202 genome from 115 other completely sequenced SARS-CoVs was also constructed. BJ202 was phylogeneticly closer to BJ01 and LLJ-2004.

Key words

severe acute respiratory syndrome associated coronavirus (SARS-CoV), genome, polymorphism