Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS

ABSTRACT One or more of the unique 3′-proximal open reading frames (ORFs) of the severe acute respiratory syndrome (SARS) coronavirus may encode determinants of virus virulence. A prime candidate is ORF6, which encodes a 63-amino-acid membrane-associated peptide that can dramatically increase the lethality of an otherwise attenuated JHM strain of murine coronavirus (L. Pewe, H. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman, J. Virol. 79:11335-11342, 2005). To discern virulence mechanisms, we compared the in vitro growth properties of rJ.6, a recombinant JHM expressing the SARS peptide, with isogenic rJ.6-KO, which has an inactive ORF containing a mutated initiation codon and a termination codon at internal position 27. The rJ.6 infections proceeded rapidly, secreting progeny about 1.5 h earlier than rJ.6-KO infections did. The rJ.6 infections were also set apart by early viral protein accumulation and by robust expansion via syncytia, a characteristic feature of JHM virus dissemination. We found no evidence for protein 6 operating at the virus entry or assembly stage, as virions from either infection were indistinguishable. Rather, protein 6 appeared to operate by fostering viral RNA and protein synthesis, as RNA quantifications by reverse transcription-quantitative PCR revealed viral RNA levels in the rJ.6 cultures that were five to eight times higher than those lacking protein 6. Furthermore, protein 6 coimmunoprecipitated with viral RNAs and colocalized on cytoplasmic vesicles with replicating viral RNAs. The SARS coronavirus encodes a novel membrane protein 6 that can accelerate replication of a related mouse virus, a property that may explain its ability to increase in vivo virus virulence.

Download Full-text

Identification of Severe Acute Respiratory Syndrome Coronavirus Replicase Products and Characterization of Papain-Like Protease Activity

Journal of Virology ◽

10.1128/jvi.78.24.13600-13612.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 13600-13612 ◽

Cited By ~ 197

Author(s):

Brian H. Harcourt ◽

Dalia Jukneliene ◽

Amornrat Kanjanahaluethai ◽

John Bechill ◽

Kari M. Severson ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Protease Activity ◽

Viral Rna ◽

Nonstructural Proteins ◽

Sars Coronavirus ◽

Hydrophobic Domain ◽

Amino Terminal ◽

Viral Proteases ◽

Infected Cells ◽

Lumenal Domain

ABSTRACT Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a ≈300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.

Download Full-text

Identification of Immunodominant Sites on the Spike Protein of Severe Acute Respiratory Syndrome (SARS) Coronavirus: Implication for Developing SARS Diagnostics and Vaccines

The Journal of Immunology ◽

10.4049/jimmunol.173.6.4050 ◽

2004 ◽

Vol 173 (6) ◽

pp. 4050-4057 ◽

Cited By ~ 89

Author(s):

Yuxian He ◽

Yusen Zhou ◽

Hao Wu ◽

Baojun Luo ◽

Jingming Chen ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Spike Protein ◽

Sars Coronavirus

Download Full-text

T-Cell Epitopes in Severe Acute Respiratory Syndrome (SARS) Coronavirus Spike Protein Elicit a Specific T-Cell Immune Response in Patients Who Recover from SARS

Journal of Virology ◽

10.1128/jvi.78.14.7861.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7861-7861 ◽

Cited By ~ 2

Author(s):

Yue-Dan Wang ◽

Wan-Yee Fion Sin ◽

Guo-Bing Xu ◽

Huang-Hao Yang ◽

Tin-yau Wong ◽

...

Keyword(s):

Immune Response ◽

T Cell ◽

Severe Acute Respiratory Syndrome ◽

Spike Protein ◽

Cell Immune Response ◽

T Cell Epitopes ◽

Sars Coronavirus

Download Full-text

Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins

Clinical and Vaccine Immunology ◽

10.1128/cvi.00351-06 ◽

2007 ◽

Vol 14 (3) ◽

pp. 331-333 ◽

Cited By ~ 14

Author(s):

Lia M. Haynes ◽

Congrong Miao ◽

Jennifer L. Harcourt ◽

Joel M. Montgomery ◽

Mai Quynh Le ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Recombinant Protein ◽

Sensitivity And Specificity ◽

Immunoglobulin G ◽

High Sensitivity ◽

Kinetic Studies ◽

Spike Protein ◽

Sars Coronavirus ◽

Nucleocapsid Proteins

ABSTRACT Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.

Download Full-text

Characterization of the Heptad Repeat Regions, HR1 and HR2, and Design of a Fusion Core Structure Model of the Spike Protein from Severe Acute Respiratory Syndrome (SARS) Coronavirus†

Biochemistry ◽

10.1021/bi049101q ◽

2004 ◽

Vol 43 (44) ◽

pp. 14064-14071 ◽

Cited By ~ 31

Author(s):

Yanhui Xu ◽

Jieqing Zhu ◽

Yiwei Liu ◽

Zhiyong Lou ◽

Fang Yuan ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Core Structure ◽

Spike Protein ◽

Heptad Repeat ◽

Structure Model ◽

Sars Coronavirus

Download Full-text

Deepening of In Silico Evaluation of SARS-CoV-2 Detection RT-qPCR Assays in the Context of New Variants

Genes ◽

10.3390/genes12040565 ◽

2021 ◽

Vol 12 (4) ◽

pp. 565

Author(s):

Mathieu Gand ◽

Kevin Vanneste ◽

Isabelle Thomas ◽

Steven Van Gucht ◽

Arnaud Capron ◽

...

Keyword(s):

Performance Evaluation ◽

Severe Acute Respiratory Syndrome ◽

In Silico ◽

The Other ◽

Viral Rna ◽

Potential Threat ◽

The World

For 1 year now, the world is undergoing a coronavirus disease-2019 (COVID-19) pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method for COVID-19 diagnosis is the detection of viral RNA by RT-qPCR with a specific set of primers and probe. It is important to frequently evaluate the performance of these tests and this can be done first by an in silico approach. Previously, we reported some mismatches between the oligonucleotides of publicly available RT-qPCR assays and SARS-CoV-2 genomes collected from GISAID and NCBI, potentially impacting proper detection of the virus. In the present study, 11 primers and probe sets investigated during the first study were evaluated again with 84,305 new SARS-CoV-2 unique genomes collected between June 2020 and January 2021. The lower inclusivity of the China CDC assay targeting the gene N has continued to decrease with new mismatches detected, whereas the other evaluated assays kept their inclusivity above 99%. Additionally, some mutations specific to new SARS-CoV-2 variants of concern were found to be located in oligonucleotide annealing sites. This might impact the strategy to be considered for future SARS-CoV-2 testing. Given the potential threat of the new variants, it is crucial to assess if they can still be correctly targeted by the primers and probes of the RT-qPCR assays. Our study highlights that considering the evolution of the virus and the emergence of new variants, an in silico (re-)evaluation should be performed on a regular basis. Ideally, this should be done for all the RT-qPCR assays employed for SARS-CoV-2 detection, including also commercial tests, although the primer and probe sequences used in these kits are rarely disclosed, which impedes independent performance evaluation.

Download Full-text

Immunological imprinting of the antibody response in COVID-19 patients

Nature Communications ◽

10.1038/s41467-021-23977-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Teresa Aydillo ◽

Alexander Rombauts ◽

Daniel Stadlbauer ◽

Sadaf Aslam ◽

Gabriela Abelenda-Alonso ◽

...

Keyword(s):

Immune Response ◽

Severe Acute Respiratory Syndrome ◽

Antibody Response ◽

Humoral Immune Response ◽

Nucleocapsid Protein ◽

Spike Protein ◽

Ongoing Research ◽

Variable Regions ◽

Humoral Immune ◽

Human Coronaviruses

AbstractIn addition to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), humans are also susceptible to six other coronaviruses, for which consecutive exposures to antigenically related and divergent seasonal coronaviruses are frequent. Despite the prevalence of COVID-19 pandemic and ongoing research, the nature of the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here we longitudinally profile the early humoral immune response against SARS-CoV-2 in hospitalized coronavirus disease 2019 (COVID-19) patients and quantify levels of pre-existing immunity to OC43, HKU1 and 229E seasonal coronaviruses, and find a strong back-boosting effect to conserved but not variable regions of OC43 and HKU1 betacoronaviruses spike protein. However, such antibody memory boost to human coronaviruses negatively correlates with the induction of IgG and IgM against SARS-CoV-2 spike and nucleocapsid protein. Our findings thus provide evidence of immunological imprinting by previous seasonal coronavirus infections that can potentially modulate the antibody profile to SARS-CoV-2 infection.

Download Full-text

Experimental Assessment of Possible Factors Associated with Tick-Borne Encephalitis Vaccine Failure

Microorganisms ◽

10.3390/microorganisms9061172 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1172

Author(s):

Ksenia Tuchynskaya ◽

Viktor Volok ◽

Victoria Illarionova ◽

Egor Okhezin ◽

Alexandra Polienko ◽

...

Keyword(s):

Vaccine Efficacy ◽

Protective Efficacy ◽

Viral Rna ◽

Dependent Manner ◽

Related Factors ◽

Effective Measure ◽

Challenge Virus ◽

Tick Borne Encephalitis ◽

Tbe Virus ◽

The Brain

Currently the only effective measure against tick-borne encephalitis (TBE) is vaccination. Despite the high efficacy of approved vaccines against TBE, rare cases of vaccine failures are well documented. Both host- and virus-related factors can account for such failures. In this work, we studied the influence of mouse strain and sex and the effects of cyclophosphamide-induced immunosuppression on the efficacy of an inactivated TBE vaccine. We also investigated how an increased proportion of non-infectious particles in the challenge TBE virus would affect the protectivity of the vaccine. The vaccine efficacy was assessed by mortality, morbidity, levels of viral RNA in the brain of surviving mice, and neutralizing antibody (NAb) titers against the vaccine strain and the challenge virus. Two-dose vaccination protected most animals against TBE symptoms and death, and protectivity depended on strain and sex of mice. Immunosuppression decreased the vaccine efficacy in a dose-dependent manner and changed the vaccine-induced NAb spectrum. The vaccination protected mice against TBE virus neuroinvasion and persistence. However, viral RNA was detected in the brain of some asymptomatic animals at 21 and 42 dpi. Challenge with TBE virus enriched with non-infectious particles led to lower NAb titers in vaccinated mice after the challenge but did not affect the protective efficacy.

Download Full-text