Javier Ortego

Generation of a Replication-Competent, Propagation-Deficient Virus Vector Based on the Transmissible Gastroenteritis Coronavirus Genome

ABSTRACT Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E+ packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-Δ3abΔE) was successfully rescued in these cell lines. rTGEV-Δ3abΔE reached high titers (107 PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 × 105 PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-Δ3abΔE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E+ packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.

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.

The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability

ABSTRACT The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg2+ ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.

Organization of Two Transmissible Gastroenteritis Coronavirus Membrane Protein Topologies within the Virion and Core

ABSTRACT The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of 35S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification.

Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway.

Abstract A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-ΔE) has been engineered. This deletion mutant only grows in cells expressing E protein (E+ cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-ΔE infected BHK-pAPN-E− cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E− cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-ΔE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-ΔE subcellular localization by confocal and immunoelectron microscopy in infected E− cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation.

Coronavirus Derived Expression Systems

Abstract Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient (>20 μg ml−1) and stable (>20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy.

The membrane M protein of the transmissible gastroenteritis coronavirus binds to the internal core through the carboxy-terminus.

An internal core was recently described in two coronaviruses, the tramsmissible gastroenteritis Coronavirus (TGEV) and the murine hepatitis virus (MHV) (Risco et al., 1996). The core has a spherical and possibly icosaedral shape. Purified cores included both the nucleoprotein and the membrane protein, as well as the viral genome. The presence of the M protein in purified cores was unexpected since this has been considered an integral membrane protein. This observation could be explained if the M protein molecules embedded in the viral envelope, with the intravirion carboxy-terminus, interact with the internal core by an intravirion domain. This hypothesis seems feasible since it was shown that the M protein interacts with the viral nucleocapsids in MHV virions (Sturman et al., 1980). However, further evidence for this interaction is required.

Nature of the Virus Associated with Endemic Balkan Nephropathy

Endemic Balkan nephropathy (EBN), a disease restricted to three Balkan countries (Bulgaria, Rumania, and Yugoslavia), is characterized by a progressive shrinking of the kidneys and, in some cases, tumors in the proximal regions of the urinary tract 1,2. A coronavirus was reported to be involved in the etiology of the disease, mostly on the basis of the isolation of a virus in cultures of kidney cells from a patient with EBN 1,3. In addition, EBN-associated virus is reported to share serologic homology with human coronaviruses OC43 and 229E, as well as the porcine transmissible gastroenteritis coronavirus (formal name: Transmissible gastroenteritis virus [TGEV]), a virus that our laboratory has been studying for 16 years 4,5. The objective of this commentary is to clarify whether the EBN-associated virus is in fact related to members of the Coronaviridae family 6.

Transmissible gastroenteritis virus (TGEV)-based vectors with engineered murine tropism express the rotavirus VP7 protein and immunize mice against rotavirus

Abstract A coronavirus vector based on the genome of the porcine transmissible gastroenteritis virus (TGEV) expressing the rotavirus VP7 protein was constructed to immunize and protect against rotavirus infections in a murine model. The tropism of this TGEV-derived vector was modified by replacing the spike S protein with the homologous protein from mouse hepatitis virus (MHV). The rotavirus gene encoding the VP7 protein was cloned into the coronavirus cDNA. BALB/c and STAT1-deficient mice were inoculated with the recombinant viral vector rTGEVS-MHV–VP7, which replicates in the intestine and spreads to other organs such as liver, spleen and lungs. TGEV-specific antibodies were detected in all the inoculated BALB/c mice, while rotavirus-specific antibodies were found only after immunization by the intraperitoneal route. Partial protection against rotavirus-induced diarrhea was achieved in suckling BALB/c mice born to dams immunized with the recombinant virus expressing VP7 when they were orally challenged with the homotypic rotavirus strain.