scholarly journals Interaction of Poly(rC) Binding Protein 2 with the 5′ Noncoding Region of Hepatitis A Virus RNA and Its Effects on Translation

1998 ◽  
Vol 72 (12) ◽  
pp. 9668-9675 ◽  
Author(s):  
Judith Graff ◽  
John Cha ◽  
Lawrence B. Blyn ◽  
Ellie Ehrenfeld
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Binding Protein ◽  
Noncoding Region ◽  
Affinity Column ◽  
Mobility Shift ◽  
Stem Loop ◽  
Rna Translation ◽  
Cell Extracts ◽  
Initiation Of Translation

ABSTRACT Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.

scholarly journals Coding Sequences Enhance Internal Initiation of Translation by Hepatitis A Virus RNA In Vitro

1998 ◽  
Vol 72 (5) ◽  
pp. 3571-3577 ◽  
Author(s):  
Judith Graff ◽  
Ellie Ehrenfeld
Keyword(s):  
Hela Cells ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Productive Infection ◽  
Initiation Codon ◽  
Translation Efficiency ◽  
Coding Sequences ◽  
Cell Extracts ◽  
Initiation Of Translation

ABSTRACT Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5′ noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5′-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5′-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5′-terminal end.

Detection of Hepatitis a Virus and other Enteroviruses in Wastewater and Surface Water Samples by Gene Probe Assay

1991 ◽  
Vol 24 (2) ◽  
pp. 267-272 ◽  
Author(s):  
S. Dubrou ◽  
H. Kopecka ◽  
J. M. Lopez Pila ◽  
J. Maréchal ◽  
J. Prévot
Keyword(s):  
Surface Water ◽  
Cell Cultures ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Limit Of Detection ◽  
Blot Hybridization ◽  
Noncoding Region ◽  
Gene Probe ◽  
Dot Blot ◽  
Lower Efficiency

Enteroviruses were specifically detected by dot blot hybridization when using poliovirus type 1 (PV1) derived subgenomic radiolabeled cRNA probes (riboprobes) in environmental water specimens and in the cell cultures in which the viruses were amplificated. The riboprobe corresponding to the 5' noncoding sequence detected the majority of enteroviruses. Hepatitis A virus (HAV) was specifically detected by an HAV cRNA probe corresponding to the 5' noncoding region of its genome. By this test, the limit of detection of coxsackievirus B5 and echovirus 7 seeded in mineral water was 103 to 104 PFU/spot. In cell cultures, positive signals were observed in the lysates of cells infected by one PFU. Higher positive signals were obtained with a short PV1 probe (nt 221-670) corresponding to the 5' noncoding region, which is a well preserved sequence among the enteroviruses, than with PV1 genomic probe. Hybridization allowed a good detection of enteroviral RNAs in wastewater specimens, but with a lower efficiency in surface water. In this case, amplification of viruses in the cell cultures gave significant hybridization results.

scholarly journals The Histone 3′-Terminal Stem-Loop-Binding Protein Enhances Translation through a Functional and Physical Interaction with Eukaryotic Initiation Factor 4G (eIF4G) and eIF3

2002 ◽  
Vol 22 (22) ◽  
pp. 7853-7867 ◽  
Author(s):  
Jun Ling ◽  
Simon J. Morley ◽  
Virginia M. Pain ◽  
William F. Marzluff ◽  
Daniel R. Gallie
Keyword(s):  
Binding Protein ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Physical Interaction ◽  
Loop Structure ◽  
Eukaryotic Initiation Factor ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Cell Extracts ◽  
Stem Loop Structure

ABSTRACT Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved stem-loop structure instead of a poly(A) tail. Not only is the stem-loop structure necessary for 3′-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone stem-loop structure is recognized by the stem-loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone stem-loop structure. Moreover, we show that the translation of mRNAs ending in the histone stem-loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5′ cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA stem-loop and the 5′ cap that is mediated by eIF4G and eIF3.

scholarly journals Detailed Analysis of the Requirements of Hepatitis A Virus Internal Ribosome Entry Segment for the Eukaryotic Initiation Factor Complex eIF4F

2001 ◽  
Vol 75 (17) ◽  
pp. 7864-7871 ◽  
Author(s):  
Andrew M. Borman ◽  
Yanne M. Michel ◽  
Katherine M. Kean
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nonstructural Protein ◽  
Binding Protein ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Internal Ribosome Entry ◽  
Eif4f Complex ◽  
Internal Ribosome Entry Segment

ABSTRACT The hepatitis A virus (HAV) internal ribosome entry segment (IRES) is unique among the picornavirus IRESs in that it is inactive in the presence of either the entero- and rhinovirus 2A or aphthovirus Lb proteinases. Since these proteinases both cleave eukaryotic initiation factor 4G (eIF4G) and HAV IRES activity could be rescued in vitro by addition of eIF4F to proteinase-treated extracts, it was concluded that the HAV IRES requires eIF4F containing intact eIF4G. Here, we show that the inability of the HAV IRES to function with cleaved eIF4G cannot be attributed to inefficient binding of the cleaved form of eIF4G by the HAV IRES. Indeed, the binding of both intact eIF4F and the C-terminal cleavage product of eIF4G to the HAV IRES was virtually indistinguishable from their binding to the encephalomyocarditis virus IRES, as assessed by UV cross-linking and filter retention assays. Rather, we show that HAV IRES activity requires, either directly or indirectly, components of the eIF4F complex which interact with the N-terminal fragment of eIF4G. Effectively, HAV IRES activity, but not that of the human rhinovirus IRES, was sensitive to the rotavirus nonstructural protein NSP3 [which displaces poly(A)-binding protein from the eIF4F complex], to recombinant eIF4E-binding protein (which prevents the association of the cap binding protein eIF4E with eIF4G), and to cap analogue.

scholarly journals 5' UTR of hepatitis A virus RNA: mutations in the 5'-most pyrimidine-rich tract reduce its ability to direct internal initiation of translation

1995 ◽  
Vol 76 (5) ◽  
pp. 1189-1196 ◽  
Author(s):  
J. S. Carneiro ◽  
M. Equestre ◽  
P. Pagnotti ◽  
A. Gradi ◽  
N. Sonenberg ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Rna ◽  
Initiation Of Translation ◽  
Internal Initiation

scholarly journals The Cys-Rich Region of Hepatitis A Virus Cellular Receptor 1 Is Required for Binding of Hepatitis A Virus and Protective Monoclonal Antibody 190/4

1998 ◽  
Vol 72 (5) ◽  
pp. 3751-3761 ◽  
Author(s):  
Peter Thompson ◽  
Jinhua Lu ◽  
Gerardo G. Kaplan
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Cell Receptor ◽  
Glycosylation Site ◽  
Immunoglobulin Superfamily ◽  
Cellular Receptor ◽  
Receptor Function ◽  
Rich Region ◽  
African Green Monkey Kidney ◽  
Cell Extracts

ABSTRACT The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1− cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1−, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.

The distance between the 3′-pyrimidine-rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA

FEBS Letters ◽  
1998 ◽  
Vol 434 (1-2) ◽  
pp. 8-12 ◽  
Author(s):  
Jane Silveira Carneiro ◽  
M Bucci ◽  
M Equestre ◽  
P Pagnotti ◽  
Alessandra Pierangeli ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Rna ◽  
Initiation Of Translation ◽  
Internal Initiation
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