Julian L. Leibowitz

Molecular and Functional Analysis of the Human Prothrombinase Gene (HFGL2) and Its Role in Viral Hepatitis

In the present studies, we report the cloning and structural characterization of the HFGL2 gene and its functional role in human fulminant hepatitis. The HFGL2 gene is approximately 7 kb in length with 2 exons. The putative promoter contains cis element consensus sequences that strongly suggest the inducibility of its expression. From the nucleotide sequence of the human gene, a 439-amino acid long protein is predicted. The overall identity between the murine fgl2 and hfgl2 coded proteins is over 70%. About 225 amino acids at the carboxyl end of these molecules are almost 90% identical, and correspond to a well-conserved fibrinogen-related domain. Both HFGL2 and FGL2 encode a type II transmembrane protein with a predicted catalytic domain toward the amino terminus of the protein. Transient transfection of Chinese hamster ovary (CHO) cells with a full-length cDNA of HFGL2 coding region resulted in high levels of prothrombinase activity. Livers from 8 patients transplanted for fulminant viral hepatitis were examined for extent of necrosis, inflammation, fibrin deposition, and HFGL2 induction. In situ hybridization showed positive staining of macrophages in areas of active hepatocellular necrosis. Fibrin stained positively in these areas and was confirmed by electron microscopy. These studies define a unique prothrombinase gene (HFGL2) and implicate its importance in the pathogenesis of fulminant viral hepatitis.

In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication

Abstract Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Δ5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells.

Murine Hepatitis Virus Strain 1 Produces a Clinically Relevant Model of Severe Acute Respiratory Syndrome in A/J Mice

ABSTRACT Severe acute respiratory syndrome (SARS) is a life-threatening infectious disease which has been difficult to study and treat because of the lack of a readily available animal model. Intranasal infection of A/J mice with the coronavirus murine hepatitis virus strain 1 (MHV-1) produced pulmonary pathological features of SARS. All MHV-1-infected A/J mice developed progressive interstitial pneumonitis, including dense macrophage infiltrates, giant cells, and hyaline membranes, resulting in death of all animals. In contrast, other mouse strains developed only mild transitory disease. Infected A/J mice had significantly higher cytokine levels, particularly macrophage chemoattractant protein 1 (MCP-1/CCL-2), gamma interferon, and tumor necrosis factor alpha. Furthermore, FGL2/fibroleukin mRNA transcripts and protein and fibrin deposits were markedly increased in the lungs of infected A/J mice. These animals developed a less robust type I interferon response to MHV-1 infection than resistant C57BL/6J mice, and treatment with recombinant beta interferon improved survival. This study describes a potentially useful small animal model of human SARS, defines its pathogenesis, and suggests treatment strategies.

Kinetic Analysis of a Unique Direct Prothrombinase, fgl2, and Identification of a Serine Residue Critical for the Prothrombinase Activity

fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-l-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent Vmax value of 6 mol/min/mol fgl2 and an apparent Km value for prothrombin of 8.3 μM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent Vmax value to 3670 mol/min/mol fgl2 and decreasing the apparent Km value for prothrombin to 7.2 μM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser89 was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.

Mitochondrial Aconitase Binds to the 3′ Untranslated Region of the Mouse Hepatitis Virus Genome

ABSTRACT Mouse hepatitis virus (MHV), a member of theCoronaviridae, contains a polyadenylated positive-sense single-stranded genomic RNA which is 31 kb long. MHV replication and transcription take place via the synthesis of negative-strand RNA intermediates from a positive-strand genomic template. Acis-acting element previously identified in the 3′ untranslated region binds to trans-acting host factors from mouse fibroblasts and forms at least three RNA-protein complexes. The largest RNA-protein complex formed by the cis-acting element and the lysate from uninfected mouse fibroblasts has a molecular weight of about 200 kDa. The complex observed in gel shift assays has been resolved by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four proteins of approximately 90, 70, 58, and 40 kDa after RNase treatment. Specific RNA affinity chromatography also has revealed the presence of a 90-kDa protein associated with RNA containing the cis-acting element bound to magnetic beads. The 90-kDa protein has been purified from uninfected mouse fibroblast crude lysates. Protein microsequencing identified the 90-kDa protein as mitochondrial aconitase. Antibody raised against purified mitochondrial aconitase recognizes the RNA-protein complex and the 90-kDa protein, which can be released from the complex by RNase digestion. Furthermore, UV cross-linking studies indicate that highly purified mitochondrial aconitase binds specifically to the MHV 3′ protein-binding element. Increasing the intracellular level of mitochondrial aconitase by iron supplementation resulted in increased RNA-binding activity in cell extracts and increased virus production as well as viral protein synthesis at early hours of infection. These results are particularly interesting in terms of identification of an RNA target for mitochondrial aconitase, which has a cytoplasmic homolog, cytoplasmic aconitase, also known as iron regulatory protein 1, a well-recognized RNA-binding protein. The binding properties of mitochondrial aconitase and the functional relevance of RNA binding appear to parallel those of cytoplasmic aconitase.

A Conserved Motif at the 3′ End of Mouse Hepatitis Virus Genomic RNA Required for Host Protein Binding and Viral RNA Replication

Abstract A conserved 11-nucleotide sequence, UGAAUGAAGUU, at the 3′ end of the genomic RNA of coronavirus mouse hepatitis virus was required for host protein binding and viral RNA synthesis. An RNA probe containing this 11-nucleotide sequence bound four cellular proteins with a highly labeled protein of 120 kDa and three minor species with sizes of 103, 81, and 55 kDa. Mutation of the 11-nucleotide motif abolished cellular protein binding. The RNA–protein complexes observed with cytoplasmic extracts from MHV-JHM-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were indistinguishable from those observed with extracts from uninfected cells. Both negative-strand synthesis and positive-strand replication of viral defective interfering RNAs in the presence of helper virus were affected by mutations that disrupt RNA–protein complex formation, even though the 11 mutated nucleotides were converted to the wild-type sequence, presumably by recombination with helper virus. Kinetic analysis indicated that recombination between DI RNA and helper virus occurred relatively early in the MHV replicative cycle at 5.5 to 7.5 hr postinfection, a time when viral RNA synthesis and replication of positive-strand DI RNA were at barely detectable levels. A DI RNA with a mutation upstream of the protein binding element replicated as efficiently as wild type without undergoing recombination. Thus, the 11-nucleotide conserved host protein binding motif appears to play an important role in viral RNA replication.

Chapter 4 - Coronavirus Pathogenesis

Abstract Coronaviruses infect many species of animals including humans, causing acute and chronic diseases. This review focuses primarily on the pathogenesis of murine coronavirus mouse hepatitis virus (MHV) and severe acute respiratory coronavirus (SARS-CoV). MHV is a collection of strains, which provide models systems for the study of viral tropism and pathogenesis in several organs systems, including the central nervous system, the liver, and the lung, and has been cited as providing one of the few animal models for the study of chronic demyelinating diseases such as multiple sclerosis. SARS-CoV emerged in the human population in China in 2002, causing a worldwide epidemic with severe morbidity and high mortality rates, particularly in older individuals. We review the pathogenesis of both viruses and the several reverse genetics systems that made much of these studies possible. We also review the functions of coronavirus proteins, structural, enzymatic, and accessory, with an emphasis on roles in pathogenesis. Structural proteins in addition to their roles in virion structure and morphogenesis also contribute significantly to viral spread in vivo and in antagonizing host cell responses. Nonstructural proteins include the small accessory proteins that are not at all conserved between MHV and SARS-CoV and the 16 conserved proteins encoded in the replicase locus, many of which have enzymatic activities in RNA metabolism or protein processing in addition to functions in antagonizing host response.

Biochemical and Genetic Analyses of Murine Hepatitis Virus Nsp15 Endoribonuclease

ABSTRACT The goal of this project was to better define the relationship between the endoribonuclease activity of murine hepatitis virus (MHV) Nsp15 (mNsp15) and its role in virus infection. Molecular modeling demonstrated that the catalytic residues of mNsp15 are superimposable with its severe acute respiratory syndrome coronavirus ortholog. Alanine substitutions at three key residues in the mNsp15 catalytic pocket (H262, H277, and G275) and a double-mutant version (H262P and H277A) generated proteins with greatly reduced but detectable endoribonuclease activities. Furthermore, these mutant proteins demonstrated lower cleavage specificities for uridylate than wild-type (WT) mNsp15. These mutations were successfully incorporated into viruses named vH262A, vH277A, vG275A, and vH262P+H277A. All four mutant viruses formed plaques with diameters similar to that of MHV-A59 1000 (WT virus) on several different cell lines. Interestingly, viruses with a mutation at a noncatalytic residue, D324A, could not be recovered despite repeated attempts, and expression of mNsp15 containing the D324A mutation in Escherichia coli resulted in an insoluble protein. Plaques derived from vH262A produced approximately 6- to 13-fold fewer PFU than those from WT virus. Cells infected with mNsp15 mutant viruses accumulated lesser amounts of plus- and minus-sense subgenomic RNAs and spike protein than WT virus. The expression of mNsp15 in trans by transient transfection partially restored RNA synthesis by vH262A. These results demonstrate that mNsp15 is required for optimal infection by MHV.

Resistance of naive mice to murine hepatitis virus strain 3 requires development of a Th1, but not a Th2, response, whereas pre-existing antibody partially protects against primary infection.

Murine hepatitis virus strain 3 (MHV-3) produces a strain-dependent spectrum of disease. The development of liver necrosis has been shown to be related to production of a unique macrophage procoagulant activity (PCA), encoded by the gene fgl-2, in susceptible mice. These studies were designed to examine the influence of Th1/Th2 cells on resistance/susceptibility and production of macrophage PCA in resistant (A/J) and susceptible (BALB/cJ) strains of mice following infection with MHV-3. Immunization of A/J mice with MHV-3 induced a Th1 cellular immune response, and one Th1 cell line (3E9.1) protected susceptible mice and inhibited PCA production by macrophages both in vitro and in vivo. In contrast, immunization of BALB/cJ mice with an attenuated variant of MHV-3 derived from passaging MHV-3 in YAC-1 cells resulted in a Th2 response. Transfer of spleen cells and T cell lines from immunized BALB/cJ mice failed to protect naive susceptible syngeneic mice from infection with MHV-3 and augmented macrophage PCA production to MHV-3 in vitro. However, serum from immunized BALB/cJ mice contained high titrated neutralizing Ab that protected naive BALB/cJ animals from lethal primary MHV-3 infection. These results demonstrate that susceptible BALB/cJ mice generate a Th2 response following MHV-3 infection and that these Th2 cells neither inhibit MHV-3-induced macrophage PCA production nor protect naive mice from MHV-3 infection. The results suggest that Ab protects against primary infection but cannot eradicate ongoing infection. Thus, these data define the differential role of Th1/Th2 lymphocytes in primary and secondary MHV-3 infection and emphasize the importance of PCA in the pathogenesis of MHV-3 infection.

Secondary Structural Elements within the 3′ Untranslated Region of Mouse Hepatitis Virus Strain JHM Genomic RNA

ABSTRACT Previously, we characterized two host protein binding elements located within the 3′-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3′-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).