Interferon-Induced Protein 44 and Interferon-Induced Protein 44-Like Restrict Replication of Respiratory Syncytial Virus.

Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defense. We have previously used a bioinformatic screen to identify two interferon-stimulated genes (ISG) with poorly characterized function, interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in respiratory syncytial virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model, we investigated the antiviral capability of these genes in the control of RSV replication. Overexpression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time postinfection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection.IMPORTANCE RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection.

Targeting human respiratory syncytial virus transcription anti-termination factor M2-1 to inhibit in vivo viral replication.

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.

Comparative study of in-situ cell death induced by the viruses of viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN) in rainbow trout.

The causative viruses of two diseases of rainbow trout, viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN), exert much of their cytopathogenic effect in cell culture through the induction of apoptosis. In the present study, the TUNEL procedure was used to investigate the presence of apoptotic cells in different organs of rainbow trout infected with the viruses of VHS and IPN. VHS viral infection resulted in massive apoptosis in renal lymphoid tissue, where viral antigens were also detected. Large numbers of viral particles were observed in close proximity to apoptotic cells. Apoptosis was not detected in excretory cells of the renal tubules or in infected muscle cells. IPN virus did not induce apoptosis in the pancreas. However, the DNA degradation associated with apoptotic nuclei was observed in muscle lesions. Taken together, these results indicated that induction of apoptosis in vivo was critically influenced by the species of virus and the cell type. Moreover, it would seem likely that apoptosis contributed to the nature of the two diseases and to mortality.

The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis.

The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.

Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway.

In this report, we show that apoptosis (or programmed cell death) is induced in different cell lines infected with a coronavirus, the porcine transmissible gastroenteritis virus (TGEV). Kinetic analysis of internucleosomal DNA cleavage by agarose gel electrophoresis and flow cytometry or cytometric monitoring of the mitochondrial transmembrane potential showed that, for ST cells infected with TGEV, the first overt signs of apoptosis appeared from 10 to 12 h postinfection on. They preceded morphological changes characteristic of cells undergoing apoptosis, as observed by light and electron microscopy. The tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked TGEV-induced apoptosis with no effect on virus production. The thiol agent pyrrolidine dithiocarbamate inhibited apoptosis, suggesting that TGEV infection may lead to apoptosis via cellular oxidative stress. The effect of TGEV infection on activation of NF-kappaB, a transcription factor known to be activated by oxidative stress, was examined. NF-kappaB DNA binding was shown to be strongly and quickly induced by TGEV infection. However, transcription factor decoy experiments showed that NF-kappaB activation is not critical for TGEV-induced apoptosis.

Complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus.

The entire nucleotide sequence of cloned cDNAs containing the 5'-untranslated region and gene 1 of Purdue-115 strain of transmissible gastroenteritis virus (TGEV) was determined. This completes the sequence of the TGEV genome, which is 28,579 nucleotides long. The gene 1 is composed of two large open reading frames, ORF1a and ORF1b, which contain 4017 and 2698 codons, respectively (stop excluded). A brief, three-codon-long ORF is present upstream of ORF1a. ORF1b overlaps ORF1a by 43 bases in the (-1) reading frame. In vitro experiments indicated that translation of the ORF1a/b polyprotein involves an efficient ribosomal frameshifting activity, as previously shown for other coronaviruses. Analysis of the predicted ORF1a and ORF1b translation products revealed that the putative functional domains identified in infectious bronchitis virus (IBV), mouse hepatitis virus (MHV) and human coronavirus 229E (HCV 229E) are all present in TGEV. The amino-terminal half of the ORF1a product exhibits greater divergence than the carboxyl-terminal half, including within the TGEV/HCV229E pair. The ORF1b protein is overall highly conserved among the above four coronaviruses, except a divergent region situated near the carboxy terminus.

Transcriptional regulation of the pyruvate kinase erythroid-specific promoter.

Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does not bind TFIID. Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites indicate that only the association of CCACC/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter. Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells.

CCACC-binding or simian-virus-40-protein-1-binding proteins cooperate with human GATA-1 to direct erythroid-specific transcription and to mediate 5' hypersensitive site 2 sensitivity of a TATA-less promoter.

Previous studies have shown that a -112 to +78 DNA fragment from the erythroid promoter of the human porphobilinogen deaminase (PBGD) gene has erythroid-specific activity. This PBGD-(-112 to +78) promoter contains a CCACC binding site (position -100), a GATA binding site (position -70) and an initiator element around the cap site. Using a cotransfection assay, we find that the human factor GATA-1 trans-activates the PBGD-(-112 to +78) promoter in non-erythroid cells. We show that, if trans-activation is abolished by mutations that destroy either the -100 CCACC binding or the -70 GATA binding sites, replacement of the -100 CCACC binding site by a simian-virus-40-protein-1 (Sp1) binding site maintains both the erythroid-specific activity of this promoter and the human GATA-1 trans-activation. Thus, human GATA-1 acts on the PBGD promoter in association with Sp1 or CCACC binding proteins. This PBGD-(-112 to +78) promoter is activated 20-fold by a cis-linked 5' hypersensitive site 2 (5'HS-2) of the human beta-globin locus control region. This activation depends on the -70 GATA and -100 CCACC or Sp1 binding sites. When a longer -714 to +78 fragment of the PBGD promoter is used, the -70 GATA mutant still displays erythroid-specific activity and is cis-activated by the 5'HS-2 enhancer, while the -100 CCACC mutant is completely inactive in the absence or in the presence of the 5'HS-2 enhancer. Thus, the -100 CCACC binding site is indispensable for the correct activity and sensitivity of the human PBGD promoter to the 5'HS-2 enhancer, whereas the -70 GATA binding site can functionally be replaced by upstream cis-acting elements.

A T-cell specific TCR delta DNA binding protein is a member of the human GATA family.

The human GATA1, hGATA1 (previously called NF-E1, GF-1 or Eryf-1), a major sequence-specific DNA-binding protein of the erythrocytic lineage, is a member of a zinc-finger family of DNA-binding proteins. We report here the cloning of a human cDNA for a new member of this family. This member, called hGATA3, has 85% amino acid homology with hGATA1 in the DNA-binding domain and no homology elsewhere in the protein. Unlike hGATA1, hGATA3 is not localized on the X chromosome and we map it to the 10p15 band of the human genome. Northern blot analysis indicates that this factor is a T-cell specific transcription factor, present before activation and up-regulated during T-cell activation. The encoded hGATA3 protein, made in an in vitro transcription-translation assay, binds the WGATAR motif present in the human T-cell receptor (TCR) delta gene enhancer and, by transfection in HeLa cells, we show that hGATA3 can transactivate this TCR delta gene enhancer. Interestingly this enhancer binds and is also transactivated by hGATA1. Conversely, the promoter of the human glycophorin B (GPB), which is erythroid-specific and contains two WGATAR motifs, binds and is transactivated by hGATA1 and, to a lesser extent, by hGATA3. These results indicate that the activation of specific genes by hGATA1 or hGATA3 is partly governed by the lineage expression of these two factors during haematopoiesis and that, in the T-cell lineage, hGATA3 binds the human TCR delta gene enhancer and is involved in its expression.

The extinction of erythroid genes after tetradecanoylphorbol acetate treatment of erythroleukemic cells correlates with down-regulation of the tissue-specific factors NF-E1 and NF-E2.

We have studied the elements involved in the tetradecanoylphorbol acetate (TPA)-mediated extinction of erythroid-specific genes. We show that transcription driven by a -714/+78-base pair DNA fragment of the erythroid promoter of the human porphobilinogen deaminase gene is down-regulated upon TPA treatment of erythroleukemic cells. Examination of the DNA binding activity of trans-acting factors involved in the expression of the porphobilinogen deaminase erythroid promoter showed (i) a constitutive expression of the CACC binding proteins and (ii) a decrease in DNA binding activity of two tissue-specific factors, NF-E1 and NF-E2. Kinetics experiments indicated that NF-E2 was down-regulated after 1 h of TPA treatment whereas NF-E1 was down-regulated at the protein and mRNA levels only after 5 h of TPA treatment. These results suggest that different pathways, acting via different transcription factors, are involved in the TPA-mediated extinction of erythroid-specific genes.

Initiation of transcription of the erythroid promoter of the porphobilinogen deaminase gene is regulated by a cis-acting sequence around the cap site.

Although the erythroid-specific promoter of human porphobilinogen deaminase [PBGD] gene has no TATA box, transcription is initiated at a single nucleotide. Using 5' and 3' deletions and point mutations, we have identified an element, located around the initiation site, which is necessary and sufficient for 'in vitro' accurate initiation of transcription. This 15 bp element extends 1 bp 5' and 14 bp 3' from the initiation site. It is composed of two regions, a proximal region centred on the cap site and a distal region which bears homology with the TdT initiator element. We show that a nuclear factor, present both in erythroid and non erythroid cells, binds the distal PBGD initiator element. Lack of heat inactivation suggests that initiation of transcription mediated by this element is not TFIID dependent. By transfection into erythroid cells, we also show that the proximal PBGD initiator element is essential for the selection of the initiation site but not for the regulation of transcription of the PBGD erythroid promoter during erythroid differentiation.

Cis- and trans-acting elements involved in the regulation of the erythroid promoter of the human porphobilinogen deaminase gene.

Two cis-acting sequences, recognized by two erythroid-specific trans-acting factors, are involved in the regulation of the erythroid promoter of the human gene coding for porphobilinogen deaminase (PBGD). The first region, located at -70, binds the erythroid factor NF-E1, and point mutations within this region abolish the induction of transcription of this promoter during murine erythroleukemia (MEL) cell differentiation. The second region, located at -160, binds the erythroid-specific factor NF-E2 and the ubiquitous factor AP1. Using UV cross-linking, we show that NF-E2 has a higher molecular weight than AP1, demonstrating that NF-E2 is not an erythroid-specific degradation product of AP1. By point mutagenesis of the NF-E2/AP1 binding site, we define mutations that abolish binding of either NF-E2 alone or AP1 and NF-E2 together. Regulation of transcription of the PBGD erythroid promoter is abolished by those mutations, suggesting that NF-E2 but not AP1 is necessary for correct regulation of this promoter in erythroid cells.