Effects of Mutating Specific Residues Present Near the Amino Terminus of 2′–5′-Oligoadenylate Synthetase

Ghosh, Arundhati; Desai, Shailesh Y.; Sarkar, Saumendra N.; Ramaraj, Pandurangan; Ghosh, Subrata; Bandyopadhyay, Smarajit; Sen, Ganes C.

doi:10.1074/jbc.272.24.15452

Cited by 31 publications

(27 citation statements)

References 20 publications

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“…6, which is published as supporting information on the PNAS web site). An N-terminal LXXXP motif is required for 2Ј-5Ј-oligoadenylate synthetase activity (30), whereas the P-loop motif is responsible for double-stranded RNA binding (31). It also has been shown that a DAD Mg 2ϩ binding motif is required for normal functioning of the murine 2Ј-5Ј-oligoadenylate synthetase (32).…”

Section: Resultsmentioning

confidence: 99%

Positional cloning of the murine flavivirus resistance gene

Perelygin

Scherbik

Zhulin

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

247

198

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Inbred mouse strains exhibit significant differences in their susceptibility to viruses in the genus Flavivirus, which includes human pathogens such as yellow fever, Dengue, and West Nile virus. A single gene, designated Flv, confers this differential susceptibility and was mapped previously to a region of mouse chromosome 5. A positional cloning strategy was used to identify 22 genes from the Flv gene interval including 10 members of the 2 -5 -oligoadenylate synthetase gene family. One 2 -5 -oligoadenylate synthetase gene, Oas1b, was identified as Flv by correlation between genotype and phenotype in nine mouse strains. Susceptible mouse strains produce a protein lacking 30% of the C-terminal sequence as compared with the resistant counterpart because of the presence of a premature stop codon. The Oas1b gene differs from all the other murine Oas genes by a unique four-amino acid deletion in the P-loop located within the conserved RNA binding domain. Expression of the resistant allele of Oas1b in susceptible embryo fibroblasts resulted in partial inhibition of the replication of a flavivirus but not of an alpha togavirus.I nnate resistance to flavivirus-induced morbidity and mortality was demonstrated in mice in the 1920s (1) and showed monogenic autosomal dominant inheritance (2). The alleles that determined resistance and susceptibility were designated Flv r and Flv s , respectively (3). Resistant mice are susceptible to infections with other types of viruses but are resistant to all flaviviruses (4). Resistant mice can be infected by flaviviruses, but the virus titers in their tissues are lower by 1,000-10,000 times than those in the tissues of susceptible animals, and the spread of the infection in resistant mice is slower (5, 6). Cell cultures derived from many different tissues of resistant mice also produce lower yields of virus; peak titers from resistant cultures are 100-1,000 times lower than those from susceptible cultures (7-9). Previous studies indicate that the Flv gene product acts intracellularly on flavivirus replication.The flavivirus-resistant allele was demonstrated in wild Mus musculus domesticus populations in both the U.S. and Australia, and flavivirus genetic resistance was reported in other Mus species (10-12). Most commonly used inbred laboratory mouse strains were derived from a small number of progenitors, and the majority of them have a homozygous flavivirus-susceptible genotype. Only the Det, BSVR, BRVR, CASA͞Rk, CAST͞Ei, MOLD͞Rk, and PRI inbred strains have the resistant allele (13). The characteristics of a resistant-like allele (designated Flv r -like) in CASA͞Rk and CAST͞Ei strains were similar to those of the PRI Flv r allele. The MOLD͞Rk animals carry an allele designated minor resistance, Flv mr , that can protect carriers from disease after infection with the attenuated 17D strain of yellow fever virus but not from the virulent Murray Valley encephalitis virus (10).The resistant allele from donor PRI mice was introduced onto the susceptible C3H͞He background to produce the co...

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Section: Resultsmentioning

confidence: 99%

Positional cloning of the murine flavivirus resistance gene

Perelygin

Scherbik

Zhulin

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

247

198

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“…Antibodies-A monoclonal antibody recognizing all small isozymes of synthetases has been described before (16). The 9-2-specific antipeptide antibody was raised in rabbit by Biosynthesis Inc.…”

Section: Methodsmentioning

confidence: 99%

A Specific Isozyme of 2′-5′ Oligoadenylate Synthetase Is a Dual Function Proapoptotic Protein of the Bcl-2 Family

Ghosh

Sarkar

Rowe

et al. 2001

Journal of Biological Chemistry

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2-5(A) synthetases are a family of interferon-induced enzymes that polymerize ATP into 2-5 linked oligoadenylates that activate RNase L and cause mRNA degradation. Because they all can synthesize 2-5(A), the reason for the existence of so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes apoptosis in mammalian cells. The proapoptotic activity of 9-2 was isozyme-specific and enzyme activity-independent. The 9-2-expressing cells exhibited many properties of cells undergoing apoptosis, such as DNA fragmentation, caspase activation, and poly ADP-ribose polymerase and lamin B cleavage. The isozyme-specific carboxyl-terminal tail of the 9-2 protein was shown, by molecular modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that it may be able to interact with members of the Bcl-2 family that contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal microscopy showed that 9-2 can indeed interact with the anti-apoptotic proteins Bcl-2 and Bclx L in vivo and in vitro. Mutations in the BH3 domain that eliminated the 9-2-Bcl-2 amd 9-2-Bclx L interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an interferon-induced dual function protein of the Bcl-2 family that can synthesize 2-5(A) and promote cellular apoptosis independently. Moreover, the cellular abundance of this protein is regulated by alternative splicing; the other isozymes encoded by the same gene are not proapoptotic.

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“…2-5 AS Assay-2-5 AS assay was done following the method described earlier (25). Briefly, 20 l of reaction mixture containing 10 g of the cell homogenate, 20 mM Tris-HCl, pH 7.5, 20 mM magnesium acetate, 2.5 mM dithiothreitol, 5 mM ATP, 5 Ci of [␣-…”

Section: Epithelial Cell Culture Virus Infection and Plaque Assay-thementioning

confidence: 99%

2′-5′ Oligoadenylate Synthetase Plays a Critical Role in Interferon-γ Inhibition of Respiratory Syncytial Virus Infection of Human Epithelial Cells

Behera

Kumar

Lockey

et al. 2002

Journal of Biological Chemistry

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Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-␥. However, the antiviral mechanism of IFN-␥ action against RSV infection is unknown. The molecular mechanism of IFN-␥-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100 -1000 units/ml of IFN-␥, either before or after RSV infection, results in a significant decrease in RSV infection. The respiratory syncytial virus (RSV)1 is the most important cause of lower respiratory tract infection in infants and young children worldwide and is a risk factor for the development of asthma. In the United States alone, RSV causes ϳ4 million cases of respiratory tract infection annually, which results in 95,000 hospitalizations and 4,500 deaths (1, 2). An effective prophylaxis or treatment against RSV is not available. Intranasal administration of a plasmid expressing IFN-␥ cDNA or a recombinant RSV expressing IFN-␥ attenuates virus replication in mice without compromising immunogenicity (3, 4). The potential and mechanism of IFN-␥ as an antiviral agent against RSV-induced lung disease remain to be elucidated.IFN-␥, a type II interferon, is a pleiotropic cytokine that plays an important role in modulating nearly all phases of immune and inflammatory responses. IFNs bind to specific receptors on cells and activate a JAK-STAT (Janus kinasesignal transducer and activator of transcription) signaling cascade that culminates in the transcriptional induction of IFNstimulated genes (ISGs) (5). The Jak1 and Jak2 phosphorylate STAT-1 following the binding of IFN-␥ to its receptor (5-7). Once phosphorylated, STAT molecules dimerize and translocate to the nucleus and bind to ␥-activated sequence elements present in the regulatory regions of various ISGs. The antiviral mechanism of IFN-␥ may involve one or more of a number of ISG-encoded products, including interferon regulatory factor-1 (IRF-1) (8), double-stranded RNA-activated protein kinase (PKR) (9, 10), the Mx family of proteins (11), a family of 2Ј-5Ј oligoadenylate synthetases (2-5 AS) (12, 13), and RNase L (14).The RNase L is constitutively expressed in most of the mammalian cells and is found in an inactive form being bound to RNase L inhibitor, RLI, a 68-kDa protein not regulated by IFN-␥ (15). The 2-5 AS produces a series of 5Ј-phosphorylated and 2Ј-and 5Ј-linked oligoadenylates (2-5A) from ATP when activated by double-stranded RNA (12, 16). It has been suggested that the binding of 2-5A with RNase L results in the release of RLI, dimerization, and the activation of RNase L (17), and subsequently activated RNase L mediates the cleavage of single-stranded RNA. However, the mechanism of the induction and activation of each of these genes vary in different cells and for the type of viruses. The mechanism of the IFN-␥-mediated antiviral activity remains to be elucidated for many clinically important viruses.Previously, we have shown that in BALB/c...

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Effects of Mutating Specific Residues Present Near the Amino Terminus of 2′–5′-Oligoadenylate Synthetase

Cited by 31 publications

References 20 publications

Positional cloning of the murine flavivirus resistance gene

Positional cloning of the murine flavivirus resistance gene

A Specific Isozyme of 2′-5′ Oligoadenylate Synthetase Is a Dual Function Proapoptotic Protein of the Bcl-2 Family

2′-5′ Oligoadenylate Synthetase Plays a Critical Role in Interferon-γ Inhibition of Respiratory Syncytial Virus Infection of Human Epithelial Cells

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