| Coronaviruses are single-stranded RNA viruses with an enveloped structure,which causes respiratory and intestinal infections in humans and animals,has previously been ignored.Since the beginning of this century,Severe Acute Respiratory Syndrome coronavirus（SARS-CoV）,Middle East Respiratory Syndrome coronavirus（MERS-CoV）and Severe Acute Respiratory Syndrome coronavirus（SARS-CoV-2）have appeared one after another.In 2002 and 2003,severe acute respiratory syndrome coronavirus（SARS-CoV）started in China and overspread in 29 countries worldwide,affecting more than 8000 people.Ten years later,MERS-CoV outbroke in Arabian Peninsula and overspread mainly in the Middle East,affecting more than 2000 people.These two outbreaks demonstrated the high transmissibility and pathogenicity of emerging coronaviruses（CoVs）.The severe acute respiratory syndrome coronavirus2（SARS-CoV-2）outbreak occurred in December 2019.The SARS‐CoV-2 has a serious impact on the world’s normal life and economy,and the world is racing to develop a vaccine to stop the spread of the epidemic.MERS-CoV is less widely spread worldwide than SARS-CoV-2,but the fatality rate is as high as 34%.According to the official report of WHO,there is no vaccine or specific treatment,and treatment is supportive and should be carried out according to the clinical condition of the patient.At present,people mainly pay attention to S protein,which will be the breakthrough of vaccine research and development.In addition to the structural protein genes such as S,the MERS-CoV coronavirus genome also encodes accessory protein genes.Interestingly,accessory protein ORF4a and ORF4b genes are located between the structural protein genes S and E.It is not clear whether this has any effect on the virus invasion.It previously reported that The MERS-CoV coronavirus accessory proteins ORF4a and ORF4b can antagonize interferon to evade the innate immune response of the host during virus invasion,thus enhancing the viral replication ability.ORF4a has been previously characterized as a dsRNA binding protein,while ORF4b is a 2’,5’-phosphodiesterase with structural and enzymatic similarity to NS2 encoded by mouse hepatitis virus（MHV）.Unfortunately,so far the structure of ORF4a and ORF4b has not been resolved.The work of this paper aims to obtain homogenous protein samples that can be used for crystallization,and to test the in vitro biological activity of the samples by various means,to provide a theoretical basis for structural analysis,and thus contribute to the development of vaccines.This paper attempts to study the structure of ORF4a and ORF4b from the perspective of structural biology.The full length ORF4a has 109 amino acids,which can exist as monomer in eukaryotic and prokaryotic expression systems.Biochemical experiments in vitro confirmed that ORF4a can bind dsRNA.Meanwhile,quantitative analysis of affinity between ORF4a and dsRNA was conducted by MST and FP methods.The full length ORF4b has 246 amino acids,and neither full-length ORF4b nor its various truncations can exist as monomers in the prokaryotic expression system.The study results showed that truncated ORF4b 53-246 existed as monomers when co-expressed with ORF4a in the eukaryotic expression system,but it’s not clear whether ORF4a is just acting as a molecular chaperone,or if there’s an interaction between the two proteins. |
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