In addition to the structural genes of the coronavirus genome, S, E, M, and N, there are several additional genes called "group-specific or accessory genes". Their gene products are designated as "accessory proteins", as reports to date make it clear that these proteins are not essential for virus replication in vitro. Nevertheless, many of these genes are still maintained in the virus genome under selective pressure, suggesting that they might play a very important role in the survival of the virus in the natural environment of the infected host. This review will summarize the research progress in the functions of coronavirus accessory genes.
Animals ; Coronavirus ; genetics ; metabolism ; Coronavirus Infections ; virology ; Humans ; Open Reading Frames ; Viral Proteins ; metabolism

Middle East respiratory syndrome (MERS) has raised global public health concerns. The recent outbreak of MERS coronavirus (MERS-CoV) infection has led to 1 338 laboratory-confirmed cases in 26 countries worldwide as reported till 19 June, 2015. MERS-CoV may be considered a zoonotic virus that has crossed the species barrier to humans, but the pathogenesis and the routes of transmission are not completely understood. Most MERS-CoV cases reported thus far have a history of residence in or travel to the Middle East. Human-to-human transmission though was observed on some occasions in Korea, it is documented as non-sustainable event. The envelope spike glycoprotein on the surface of MERS-CoV which mediates receptor binding, membrane fusion and viral entry is thought to be involved in the mechanism of MERS-CoV.No specific and effective treatment for MERS-CoV is currently recommended, although supportive treatment has played an important role. Prophylactic strategies are necessary to prevent MERS-CoV infection.
Coronavirus Infections ; diagnosis ; epidemiology ; therapy ; Humans ; Middle East Respiratory Syndrome Coronavirus ; pathogenicity ; physiology ; Spike Glycoprotein, Coronavirus ; metabolism ; Virus Internalization

In 2012, a new SARS-like coronavirus emerged in the Middle East, namely the Middle East respiratory syndrome coronavirus (MERS-CoV). It has caused outbreaks with high mortality. During infection of target cell, MERS-CoV S protein S1 subunit binds to the cellular receptor (DPP4), and its S2 subunit HR1 and HR2 regions intact with each other to form a stable six-helix bundle to mediate the fusion between virus and target cell membranes. Hence, blocking the process of six-helix bundle formation can effectively inhibit MERS-CoV entry into the target cells. This review focuses on the recent advance in the development of peptidic entry inhibitors targeting the MERS-CoV S2 subunit.
Antiviral Agents ; pharmacology ; Coronavirus Infections ; drug therapy ; Dipeptidyl Peptidase 4 ; metabolism ; Drug Design ; Humans ; Middle East Respiratory Syndrome Coronavirus ; drug effects ; physiology ; Peptides ; pharmacology ; Spike Glycoprotein, Coronavirus ; metabolism ; Virus Internalization ; drug effects

Amino Acid Sequence ; Coronavirus/drug effects* ; Coronavirus Infections/drug therapy* ; Drug Design ; Humans ; Protein Structure, Secondary ; Spike Glycoprotein, Coronavirus/metabolism* ; Viral Fusion Protein Inhibitors/pharmacology* ; Viral Fusion Proteins/metabolism*

Porcine epidemic diarrhea virus (PEDV) causes an acute enteritis in pigs of all ages, often fatality for neonates. PEDV occupies an intermediate position between two well characterized members of the coronavirus group I, human coronavirus (HCoV-229E)and transmissible gastroenteritis virus (TGEV) which uses aminopeptidase N (APN), a 150 kDa protein, as their receptors. However, the receptor of the PEDV has not been identified yet. A virus overlay protein binding assay (VOPBA) was used to identify PEDV binding protein in permissive cells. The binding ability of PEDV to porcine APN (pAPN) and the effects of pAPN on infectivity of PEDV in Vero cells were also investigated. VOPBA identified a 150 kDa protein, as a putative PEDV receptor in enterocytes and swine testicle (ST) cells. Further the PEDV binding to pAPN was blocked by anti-pAPN and pAPN enhanced PEDV infectivity in Vero cells. In conclusion, these results suggested that pAPN may act as a receptor of PEDV.
Animals ; Antigens, CD13/*metabolism ; Cercopithecus aethiops ; Coronavirus/*metabolism ; Coronavirus Infections/*veterinary/virology ; Digestive System Diseases/metabolism/*veterinary/virology ; Enterocytes/enzymology/metabolism/virology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Male ; Protein Binding ; Receptors, Virus/*metabolism ; Swine ; Swine Diseases/metabolism/*virology ; Vero Cells

No abstract available.
Coronavirus Infections/*diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; Nasopharynx/virology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.
Animals ; Coronavirus Infections ; metabolism ; virology ; Coronavirus NL63, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Mice ; Mice, Inbred BALB C ; Protein Engineering ; Protein Structure, Tertiary ; Receptors, Virus ; metabolism ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (M(pro)) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. M(pro) is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, M(pro) can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature M(pro) self-assembles into a novel super-active octamer (AO-M(pro)). The crystal structure of AO-M(pro) adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported M(pro) active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of M(pro), in equilibrium between inactive monomer and active dimer, the stable AO-M(pro) exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-M(pro) and its polyprotein substrates with multiple cleavage sites, would make AO-M(pro) functionally much more superior than the M(pro) active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AOM(pro) should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AOM(pro) provides new insights about the functional mechanism of M(pro) and its maturation process.
Coronavirus ; metabolism ; Cysteine Endopeptidases ; Endopeptidases ; metabolism ; Humans ; Peptides ; chemistry ; metabolism ; Polyproteins ; chemistry ; metabolism ; Protein Binding ; SARS Virus ; chemistry ; metabolism ; Viral Proteins

BACKGROUND: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. METHODS: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). RESULTS: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.
Acetylcysteine/chemistry ; Citrates/chemistry ; Coronavirus Infections/diagnosis ; Deoxyribonuclease I/metabolism ; Endopeptidase K/metabolism ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/*isolation & purification ; RNA, Viral/analysis/*isolation & purification/metabolism ; Real-Time Polymerase Chain Reaction ; Sputum/*virology

To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus (IBV) Beaudette strain, the ectodomain region of the Spike gene (1,302 bp) of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA. This recombinant was designated as BeauR-H120(S1). BeauR-H120(S1) was directly used as the DNA template for the transcription of viral genomic RNA in vitro. Then, the transcription product was transfected into Vero cells by electroporation. At 48 h post-transfection, the transfected Vero cells were harvested, and passaging continued. A syncytium was not observed until the recombinant virus had passed through four passages. The presence of rBeau-H120(S1) was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR. Western blot analysis of rBeau-H120 (S1)-infected Vero cell lysates demonstrated that the nucleocapsid (N) protein was expressed, which implied that rBeau-H120(S1) could propagate in Vero cells. The TCIDs0 and EIDs0 data demonstrated that the titer levels of rBeau-H120(S1) reached 10(590+/-0.22)TCID50/mL and 10(6.13+/-0.23)EID50/mL in Vero cells and 9-day-old SPF chicken embryos, respectively. Protection studies showed that the percentage of antibody-positive chickens, which were vaccinated with rBeau-H120(S1) at 7 days after hatching, rose to 90% at 21 days post-inoculation. Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41 strain (103EID50/chicken). This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis.
Animals ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Coronavirus Infections ; veterinary ; virology ; Infectious bronchitis virus ; chemistry ; genetics ; growth & development ; metabolism ; Poultry Diseases ; virology ; Protein Structure, Tertiary ; Spike Glycoprotein, Coronavirus ; chemistry ; genetics ; metabolism ; Transfection ; Vero Cells