BiP Internal Ribosomal Entry Site Activity Is Controlled by Heat-Induced Interaction of NSAP1

ABSTRACT TheBiP protein, a stress response protein, plays an important role in the proper folding and assembly of nascent protein and in the scavenging of misfolded proteins in the endoplasmic reticulum lumen. Translation of BiP is directed by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of the BiP mRNA. BiP IRES activity increases when cells are heat stressed. Here we report that NSAP1 specifically enhances the IRES activity of BiP mRNA by interacting with the IRES element. Overexpression of NSAP1 in 293T cells increased the IRES activity of BiP mRNA, whereas knockdown of NSAP1 by small interfering RNA (siRNA) reduced the IRES activity of BiP mRNA. The amount of NSAP1 bound to the BiP IRES increased under heat stress conditions, and the IRES activity of BiP mRNA was increased. Moreover, the increase in BiP IRES activity with heat treatment was not observed in cells lacking NSAP1 after siRNA treatment. BiP mRNAs were redistributed from the heavy polysome to the light polysome in NSAP1 knockdown cells. Together, these data indicate that NSAP1 modulates IRES-dependent translation of BiP mRNA through an RNA-protein interaction under heat stress conditions.

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Dual Stem Loops within the Poliovirus Internal Ribosomal Entry Site Control Neurovirulence

Journal of Virology ◽

10.1128/jvi.73.2.958-964.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 958-964 ◽

Cited By ~ 100

Author(s):

Matthias Gromeier ◽

Birgit Bossert ◽

Mineo Arita ◽

Akio Nomoto ◽

Eckard Wimmer

Keyword(s):

Internal Ribosomal Entry Site ◽

Internal Constraints ◽

Virus Propagation ◽

Entry Site ◽

Stem Loop ◽

Nontranslated Region ◽

Growth Properties ◽

Ribosomal Entry ◽

Site Control

ABSTRACT In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5′ nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation intoCynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.

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La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site

Journal of General Virology ◽

10.1099/0022-1317-80-12-3159 ◽

1999 ◽

Vol 80 (12) ◽

pp. 3159-3166 ◽

Cited By ~ 33

Author(s):

Yoon Ki Kim ◽

Sung Key Jang

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein A ◽

Internal Ribosomal Entry Site ◽

Encephalomyocarditis Virus ◽

Entry Site ◽

Polypyrimidine Tract Binding Protein ◽

La Protein ◽

Ribosomal Entry

Translation of internal ribosomal entry site (IRES)-dependent mRNAs is mediated by RNA-binding proteins as well as canonical translation factors. In order to elucidate the roles of RNA-binding proteins in IRES-dependent translation, the role of polypyrimidine tract-binding protein (PTB) and La protein in encephalomyocarditis virus (EMCV) IRES-dependent translation was investigated. PTB was required for efficient EMCV IRES-driven translation but, intriguingly, an excess of PTB suppressed it. Such a translational suppression by surplus PTB was relieved by addition of La protein. A possible role for La protein in IRES-dependent translation is discussed.

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Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3' nontranslated region

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2436464100 ◽

2003 ◽

Vol 100 (25) ◽

pp. 15125-15130 ◽

Cited By ~ 42

Author(s):

E. Dobrikova ◽

P. Florez ◽

S. Bradrick ◽

M. Gromeier

Keyword(s):

Internal Ribosomal Entry Site ◽

Entry Site ◽

Nontranslated Region ◽

Ribosomal Entry

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Replication of Poliovirus Requires Binding of the Poly(rC) Binding Protein to the Cloverleaf as Well as to the Adjacent C-Rich Spacer Sequence between the Cloverleaf and the Internal Ribosomal Entry Site

Journal of Virology ◽

10.1128/jvi.00516-07 ◽

2007 ◽

Vol 81 (18) ◽

pp. 10017-10028 ◽

Cited By ~ 54

Author(s):

Hidemi Toyoda ◽

David Franco ◽

Kentaro Fujita ◽

Aniko V. Paul ◽

Eckard Wimmer

Keyword(s):

Binding Protein ◽

Position Effect ◽

Internal Ribosomal Entry Site ◽

Rna Replication ◽

Cellular Protein ◽

Entry Site ◽

Stem Loop ◽

Nontranslated Region ◽

Ribosomal Entry

ABSTRACT The 5′ nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CDpro. The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C93-95 and C98-100) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C93-95) resulted in slower viral growth, whereas the C98-100A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C94-95) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5′-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CDpro to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.

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