Isabel Sola

Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis

ABSTRACT Coronavirus transcription leads to the synthesis of a nested set of mRNAs with a leader sequence derived from the 5′ end of the genome. The mRNAs are produced by a discontinuous transcription in which the leader is linked to the mRNA coding sequences. This process is regulated by transcription-regulating sequences (TRSs) preceding each mRNA, including a highly conserved core sequence (CS) with high identity to sequences present in the virus genome and at the 3′ end of the leader (TRS-L). The role of TRSs was analyzed by reverse genetics using a full-length infectious coronavirus cDNA and site-directed mutagenesis of the CS. The canonical CS-B was nonessential for the generation of subgenomic mRNAs (sgmRNAs), but its presence led to transcription levels at least 103-fold higher than those in its absence. The data obtained are compatible with a transcription mechanism including three steps: (i) formation of 5′-3′ complexes in the genomic RNA, (ii) base-pairing scanning of the nascent negative RNA strand by the TRS-L, and (iii) template switching during synthesis of the negative strand to complete the negative sgRNA. This template switch takes place after copying the CS sequence and was predicted in silico based on high base-pairing score between the nascent negative RNA strand and the TRS-L and minimum ΔG.

Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection. Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

ABSTRACT The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2′-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence

Abstract Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3′-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5′ end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-Δ7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-Δ7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

Coronavirus nucleocapsid protein is an RNA chaperone.

Abstract RNA chaperones are nonspecific nucleic acid binding proteins with long disordered regions that help RNA molecules to adopt its functional conformation. Coronavirus nucleoproteins (N) are nonspecific RNA-binding proteins with long disordered regions. Therefore, we investigated whether transmissible gastroenteritis coronavirus (TGEV) N protein was an RNA chaperone. Purified N protein enhanced hammerhead ribozyme self-cleavage and nucleic acids annealing, which are properties that define RNA chaperones. In contrast, another RNA-binding protein, PTB, did not show these activities. N protein chaperone activity was blocked by specific monoclonal antibodies. Therefore, it was concluded that TGEV N protein is an RNA chaperone. In addition, we have shown that purified severe acute respiratory syndrome (SARS)-CoV N protein also has RNA chaperone activity. In silico predictions of disordered domains showed a similar pattern for all coronavirus N proteins evaluated. Altogether, these data led us to suggest that all coronavirus N proteins might be RNA chaperones.

Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk.

Protection against enteric infections can be provided by the oral administration of pathogen-neutralizing antibodies. To provide passive immunity, 18 lines of transgenic mice secreting a recombinant monoclonal antibody (Mab) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. The genes encoding a chimeric Mab with the variable modules of the murine TGEV-specific Mab 6A.C3 and the constant modules of a human IgG1, isotype Mab were expressed under the control of regulatory sequences derived from the whey acidic protein, which is an abundant milk protein. The Mab 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species, which does not allow the selection of neutralization escape mutants. Antibody expression titers of 106 were obtained in the milk of transgenic mice that reduced TGEV infectivity 106-fold. The antibody was synthesized at high levels throughout lactation. Integration of matrix attachment region sequences with the antibody genes led to a 20- to 10,000-fold increase in the antibody titer in 50% of the transgenic animals. Antibody expression levels were transgene copy number independent and related to the site of integration. The generation of transgenic animals producing virus neutralizing antibodies in milk could provide an approach to protection against neonatal infections of the enteric tract.

Complete genome sequence of transmissible gastroenteritis coronavirus PUR46-MAD clone and evolution of the purdue virus cluster.

The complete sequence (28580 nt) of the PUR46-MAD clone of the Purdue cluster of transmissible gastroenteritis coronavirus (TGEV) has been determined and compared with members of this cluster and other coronaviruses. The computing distances among their S gene sequences resulted in the grouping of these coronaviruses into four clusters, one of them exclusively formed by the Purdue viruses. Three new potential sequence motifs with homology to the α-subunit of the polymerase-associated nucleocapsid phosphoprotein of rinderpest virus, the Bowman–Birk type of proteinase inhibitors, and the metallothionein superfamily of cysteine rich chelating proteins have been identified. Comparison of the TGEV polymerase sequence with that of other RNA viruses revealed high sequence homology with the A–E domains of the palm subdomain of nucleic acid polymerases.

Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity.

ABSTRACT The genome of the coronavirus transmissible gastroenteritis virus (TGEV) has been engineered as an expression vector with an infectious cDNA. The vector led to the efficient (>40 μg/106 cells) and stable (>20 passages) expression of a heterologous gene (green fluorescent protein [GFP]), driven by the transcription-regulating sequences (TRS) of open reading frame (ORF) 3a inserted in the site previously occupied by the nonessential ORFs 3a and 3b. Expression levels driven by this TRS were higher than those of an expression cassette under the control of regulating sequences engineered with the N gene TRS. The recombinant TGEV including the GFP gene was still enteropathogenic, albeit with a 10- to 102-fold reduction in enteric tissue growth. Interestingly, a specific lactogenic immune response against the heterologous protein has been elicited in sows and their progeny. The engineering of an additional insertion site for the heterologous gene between viral genes N and 7 led to instability and to a new genetic organization of the 3′ end of the recombinant viruses. As a consequence, a major species of subgenomic mRNA was generated from a TRS with the noncanonical core sequence 5′-CUAAAA-3′. Extension of the complementarity between the TRS and sequences at the 3′ end of the viral leader was associated with transcriptional activation of noncanonical core sequences. The engineered vector led to expression levels as high as those of well-established vectors and seems very promising for the development of vaccines and, possibly, for gene therapy.

Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants of Escherichia coli Cultures by Use of the Hemolysin System

ABSTRACT A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants ofEscherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFvs can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

Role of Nucleotides Immediately Flanking the Transcription-Regulating Sequence Core in Coronavirus Subgenomic mRNA Synthesis

ABSTRACT The generation of subgenomic mRNAs in coronavirus involves a discontinuous mechanism of transcription by which the common leader sequence, derived from the genome 5′ terminus, is fused to the 5′ end of the mRNA coding sequence (body). Transcription-regulating sequences (TRSs) precede each gene and include a conserved core sequence (CS) surrounded by relatively variable sequences (5′ TRS and 3′ TRS). Regulation of transcription in coronaviruses has been studied by reverse-genetics analysis of the sequences immediately flanking a unique CS in the Transmissible gastroenteritis virus genome (CS-S2), located inside the S gene, that does not lead to detectable amounts of the corresponding mRNA, in spite of its canonical sequence. The transcriptional inactivity of CS-S2 was genome position independent. The presence of a canonical CS was not sufficient to drive transcription, but subgenomic synthesis requires a minimum base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) provided by the CS and its adjacent nucleotides. A good correlation was observed between the free energy of TRS-L and cTRS-B duplex formation and the levels of subgenomic mRNA S2, demonstrating that base pairing between the leader and body beyond the CS is a determinant regulation factor in coronavirus transcription. In TRS mutants with increasing complementarity between TRS-L and cTRS-B, a tendency to reach a plateau in ΔG values was observed, suggesting that a more precise definition of the TRS limits might be proposed, specifically that it consists of the central CS and around 4 nucleotides flanking 5′ and 3′ the CS. Sequences downstream of the CS exert a stronger influence on the template-switching decision according to a model of polymerase strand transfer and template switching during minus-strand synthesis.