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

Virology ◽  
1995 ◽  
Vol 214 (1) ◽  
pp. 128-138 ◽  
Author(s):  
WEI YU ◽  
JULIAN L. LEIBOWITZ
Keyword(s):  
Protein Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Genomic Rna ◽  
Conserved Motif

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

2001 ◽  
Vol 75 (24) ◽  
pp. 12105-12113 ◽  
Author(s):  
Qi Liu ◽  
Reed F. Johnson ◽  
Julian L. Leibowitz
Keyword(s):  
Secondary Structure ◽  
Protein Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Replication ◽  
Secondary Structures ◽  
Host Protein ◽  
Wild Type ◽  
Base Pairing ◽  
Stem Loop

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).

scholarly journals Effect of Mutations in the Mouse Hepatitis Virus 3′(+)42 Protein Binding Element on RNA Replication

2005 ◽  
Vol 79 (23) ◽  
pp. 14570-14585 ◽  
Author(s):  
Reed F. Johnson ◽  
Min Feng ◽  
Pinghua Liu ◽  
Jason J. Millership ◽  
Boyd Yount ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Replication ◽  
Host Protein ◽  
Reverse Genetic System ◽  
Wild Type ◽  
Mobility Shift ◽  
Ribonucleoprotein Complexes

ABSTRACT The mouse hepatitis virus (MHV) genome's 3′ untranslated region contains cis-acting sequences necessary for replication. Studies of MHV and other coronaviruses have indicated a role for RNA secondary and tertiary elements in replication. Previous work in our laboratory has identified four proteins which form ribonucleoprotein complexes with the 3′-terminal 42 nucleotides [3′(+)42] of the MHV genome. Defective interfering (DI) RNA replication assays have demonstrated a role for the 3′(+)42 host protein binding element in the MHV life cycle. Using gel mobility shift RNase T1 protection assays and secondary structure modeling, we have characterized a possible role for RNA secondary structure in host protein binding to the 3′-terminal 42-nucleotide element. Additionally we have identified a role for the 3′-terminal 42-nucleotide host protein binding element in RNA replication and transcription using DI RNA replication assays and targeted recombination and by directly constructing mutants in this protein binding element using a recently described MHV reverse genetic system. DI RNA replication assays demonstrated that mutations in the 3′(+)42 host protein binding element had a deleterious effect on the accumulation of DI RNA. When the identical mutations were directly inserted into the MHV genome, most mutant genomes were viable but formed smaller plaques than the wild-type parent virus. One mutant was not viable. This mutant directed the synthesis of genome-sized negative-sense RNA approximately as efficiently as the wild type did but had a defect in subgenomic mRNA synthesis. These results point to a potential role for sequences at the extreme 3′ end of the MHV genome in subgenomic RNA synthesis.

scholarly journals RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

2002 ◽  
Vol 76 (8) ◽  
pp. 3697-3708 ◽  
Author(s):  
Rainer Gosert ◽  
Amornrat Kanjanahaluethai ◽  
Denise Egger ◽  
Kurt Bienz ◽  
Susan C. Baker
Keyword(s):  
Electron Microscopy ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Membrane Vesicles ◽  
Rna Replication ◽  
Viral Rna ◽  
Double Membrane ◽  
Ultrastructural Studies ◽  
Infected Cells

ABSTRACT The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5′-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

scholarly journals Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12008-12022 ◽  
Author(s):  
Brandon L. Walter ◽  
Todd B. Parsley ◽  
Ellie Ehrenfeld ◽  
Bert L. Semler
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

scholarly journals Yeast Lsm1p-7p/Pat1p Deadenylation-Dependent mRNA-Decapping Factors Are Required for Brome Mosaic Virus Genomic RNA Translation

2003 ◽  
Vol 23 (12) ◽  
pp. 4094-4106 ◽  
Author(s):  
Amine O. Noueiry ◽  
Juana Diez ◽  
Shaun P. Falk ◽  
Jianbo Chen ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Genomic Rna ◽  
Reading Frame ◽  
Mrna Decapping ◽  
Rna Translation ◽  
Positive Strand Rna

ABSTRACT Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5′ and 3′ noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.

scholarly journals Sensitivity of Alphaviruses to G3BP Deletion Correlates with Efficiency of Replicase Polyprotein Processing

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Benjamin Götte ◽  
Age Utt ◽  
Rennos Fragkoudis ◽  
Andres Merits ◽  
Gerald M. McInerney
Keyword(s):  
Chikungunya Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Regulatory Role ◽  
Functional Link ◽  
Ongoing Research ◽  
Old World ◽  
Polyprotein Processing ◽  
Viral Replicase

ABSTRACT We present a comprehensive overview of the dependency of several Old World alphaviruses for the host protein G3BP. Based on their replication ability in G3BP-deleted cells, Old World alphaviruses can be categorized into two groups, being either resistant or sensitive to G3BP deletion. We observed that all sensitive viruses have an Arg residue at the P4 position of the cleavage site between the nonstructural protein P1 (nsP1) and nsP2 regions of the replicase precursor polyprotein (1/2 site), while a different residue is found at this site in viruses resistant to G3BP deletion. Swapping this residue between resistant and sensitive viruses also switches the G3BP deletion sensitivity. In the absence of G3BP, chikungunya virus (CHIKV) replication is at the limit of detection. The P4 Arg-to-His substitution partially rescues this defect. The P4 residue of the 1/2 site is known to play a regulatory role during processing at this site, and we found that if processing is blocked, the influence of the P4 residue on the sensitivity to G3BP deletion is abolished. Immunofluorescence experiments with CHIKV replicase with manipulated processing indicate that the synthesis of double-stranded RNA is defective in the absence of G3BP and suggest a role of G3BP during negative-strand RNA synthesis. This study provides a functional link between the host protein G3BP and the P4 residue of the 1/2 site for viral RNA replication of Old World alphaviruses. While this suggests a link between G3BP proteins and viral replicase polyprotein processing, we propose that G3BP proteins do not have a regulatory role during polyprotein processing. IMPORTANCE Old World alphaviruses comprise several medically relevant viruses, including chikungunya virus and Ross River virus. Recurrent outbreaks and the lack of antivirals and vaccines demand ongoing research to fight the emergence of these infectious diseases. In this context, a thorough investigation of virus-host interactions is critical. Here, we highlight the importance of the host protein G3BP for several Old World alphaviruses. Our data strongly suggest that G3BP plays a crucial role for the activity of the viral replicase and, thus, the amplification of the viral RNA genome. To our knowledge, the present work is the first to provide a functional link between the regulation of viral polyprotein processing and RNA replication and a host factor for alphaviruses. Moreover, the results of this study raise several questions about the fundamental regulatory mechanisms that dictate the activity of the viral replicase, thereby paving the way for future studies.

scholarly journals Modification of picornavirus genomic RNA using ‘click’ chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

2013 ◽  
Vol 42 (4) ◽  
pp. 2473-2482 ◽  
Author(s):  
Martijn A. Langereis ◽  
Qian Feng ◽  
Frank H. T. Nelissen ◽  
Richard Virgen-Slane ◽  
Gerbrand J. van der Heden van Noort ◽  
...  
Keyword(s):  
Click Reaction ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Viral Rna ◽  
Host Protein ◽  
Genomic Rna ◽  
Rna Molecules ◽  
Rna Translation ◽  
Mouth Disease Virus ◽  
Covalently Linked

Abstract Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formed viral RNA molecules possess a covalently linked VPg peptide. It is known that VPg is enzymatically released from the incoming viral RNA by a host protein, called TDP2, but it is still unclear whether the release of VPg is necessary to initiate RNA translation. To study the possible requirement of VPg release for RNA translation, we developed a novel method to modify the genomic viral RNA with VPg linked via a ‘non-cleavable’ bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne [bicyclo[6.1.0]nonyne (BCN)] by a copper-free ‘click’ reaction, leading to a VPg-triazole-RNA construct that was ‘non-cleavable’ by TDP2. We successfully ligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We show that the lack of VPg unlinkase does not influence RNA translation or replication. Thus, the release of the VPg from the incoming viral RNA is not a prerequisite for RNA translation or replication.

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