An Amphipathic α-Helix Controls Multiple Roles of Brome Mosaic Virus Protein 1a in RNA Replication Complex Assembly and Function

ABSTRACT Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.

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Brome Mosaic Virus Protein 1a Recruits Viral RNA2 to RNA Replication through a 5′ Proximal RNA2 Signal

Journal of Virology ◽

10.1128/jvi.75.7.3207-3219.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3207-3219 ◽

Cited By ~ 91

Author(s):

Jianbo Chen ◽

Amine Noueiry ◽

Paul Ahlquist

Keyword(s):

Mosaic Virus ◽

Rna Virus ◽

Rna Replication ◽

Brome Mosaic Virus ◽

Virus Protein ◽

Membrane Association ◽

Stem Loop ◽

Induced Membrane ◽

Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5′-proximal third of RNA2. The RNA2 5′ untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5′-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TΨC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

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Brome Mosaic Virus RNA Replication Proteins 1a and 2a Colocalize and 1a Independently Localizes on the Yeast Endoplasmic Reticulum

Journal of Virology ◽

10.1128/jvi.73.12.10303-10309.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10303-10309 ◽

Cited By ~ 98

Author(s):

María Restrepo-Hartwig ◽

Paul Ahlquist

Keyword(s):

Endoplasmic Reticulum ◽

Mosaic Virus ◽

Plant Cells ◽

Rna Replication ◽

Brome Mosaic Virus ◽

Viral Rna ◽

Replication Proteins ◽

Replication Complex ◽

Virus Rna ◽

Positive Strand Rna

ABSTRACT The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeast Saccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined. For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting. As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus. 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER. In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis. In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes. Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA replication complex.

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The Heat Shock Protein 70 Cochaperone YDJ1 Is Required for Efficient Membrane-Specific Flock House Virus RNA Replication Complex Assembly and Function in Saccharomyces cerevisiae

Journal of Virology ◽

10.1128/jvi.02017-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 2004-2012 ◽

Cited By ~ 27

Author(s):

Spencer A. Weeks ◽

David J. Miller

Keyword(s):

Rna Replication ◽

Viral Rna ◽

Flock House Virus ◽

Complex Activity ◽

Complex Assembly ◽

Replication Complex ◽

Hsp90 Chaperone ◽

Chaperone Complex ◽

And Function

ABSTRACT The assembly of RNA replication complexes on intracellular membranes is an essential step in the life cycle of positive-sense RNA viruses. We have previously shown that Hsp90 chaperone complex activity is essential for efficient Flock House virus (FHV) RNA replication in Drosophila melanogaster S2 cells. To further explore the role of cellular chaperones in viral RNA replication, we used both pharmacologic and genetic approaches to examine the role of the Hsp90 and Hsp70 chaperone systems in FHV RNA replication complex assembly and function in Saccharomyces cerevisiae. In contrast to results with insect cells, yeast deficient in Hsp90 chaperone complex activity showed no significant decrease in FHV RNA replication. However, yeast with a deletion of the Hsp70 cochaperone YDJ1 showed a dramatic reduction in FHV RNA replication that was due in part to reduced viral RNA polymerase accumulation. Furthermore, the absence of YDJ1 did not reduce FHV RNA replication when the viral RNA polymerase and replication complexes were retargeted from the mitochondria to the endoplasmic reticulum. These results identify YDJ1 as an essential membrane-specific host factor for FHV RNA replication complex assembly and function in S. cerevisiae and are consistent with known differences in the role of distinct chaperone complexes in organelle-specific protein targeting between yeast and higher eukaryotes.

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An Alternate Pathway for Recruiting Template RNA to the Brome Mosaic Virus RNA Replication Complex

Journal of Virology ◽

10.1128/jvi.77.4.2568-2577.2003 ◽

2003 ◽

Vol 77 (4) ◽

pp. 2568-2577 ◽

Cited By ~ 34

Author(s):

Jianbo Chen ◽

Amine Noueiry ◽

Paul Ahlquist

Keyword(s):

Mosaic Virus ◽

Rna Replication ◽

Brome Mosaic Virus ◽

Terminal Region ◽

Open Reading Frame ◽

Viral Rna ◽

Stem Loop ◽

Reading Frame ◽

Replication Complex ◽

Alternate Pathway

ABSTRACT The multidomain RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, plays key roles in assembly and function of the viral RNA replication complex. 1a, which encodes RNA capping and helicase-like domains, localizes to endoplasmic reticulum membranes, recruits BMV 2a polymerase and viral RNA templates, and forms membrane-bound, capsid-like spherules in which RNA replication occurs. cis-acting signals necessary and sufficient for RNA recruitment by 1a have been mapped in BMV genomic RNA2 and RNA3. Both signals comprise an extended stem-loop whose apex matches the conserved sequence and structure of the TΨC stem-loop in tRNAs (box B). Mutations show that this box B motif is crucial to 1a responsiveness of wild-type RNA2 and RNA3. We report here that, unexpectedly, some chimeric mRNAs expressing the 2a polymerase open reading frame from RNA2 were recruited by 1a to the replication complex and served as templates for negative-strand RNA synthesis, despite lacking the normally essential, box B-containing 5′ signal. Further studies showed that this template recruitment required high-efficiency translation of the RNA templates. Moreover, multiple small frameshifting insertion or deletion mutations throughout the N-terminal region of the open reading frame inhibited this template recruitment, while an in-frame insertion did not. Providing 2a in trans did not restore template recruitment of RNAs with frameshift mutations. Only those deletions in the N-terminal region of 2a that abolished 2a interaction with 1a abolished template recruitment of the RNA. These and other results indicate that this alternate pathway for 1a-dependent RNA recruitment involves 1a interaction with the translating mRNA via the 1a-interactive N-terminal region of the nascent 2a polypeptide. Interaction with nascent 2a also may be involved in 1a recruitment of 2a polymerase to membranes.

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Brome Mosaic Virus Polymerase-Like Protein 2a Is Directed to the Endoplasmic Reticulum by Helicase-Like Viral Protein 1a

Journal of Virology ◽

10.1128/jvi.74.9.4310-4318.2000 ◽

2000 ◽

Vol 74 (9) ◽

pp. 4310-4318 ◽

Cited By ~ 110

Author(s):

Jianbo Chen ◽

Paul Ahlquist

Keyword(s):

Endoplasmic Reticulum ◽

Mosaic Virus ◽

Rna Replication ◽

Brome Mosaic Virus ◽

Viral Rna ◽

Yeast Cells ◽

Cell Fractionation ◽

Cytoplasmic Organelle ◽

Replication Complex ◽

Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a. 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV-specific RNA-dependent RNA synthesis in plant and yeast cells. 1a also localizes to the ER in the absence of 2a or viral RNA replication templates. To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription. In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested. Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a. When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus. Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction. Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER. These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly.

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

Molecular and Cellular Biology ◽

10.1128/mcb.23.12.4094-4106.2003 ◽

2003 ◽

Vol 23 (12) ◽

pp. 4094-4106 ◽

Cited By ~ 70

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.

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Brome Mosaic Virus RNA Replication and Transcription

Viral Genome Replication ◽

10.1007/b135974_5 ◽

2009 ◽

pp. 89-108

Author(s):

Guanghui Yi ◽

C. Cheng Kao

Keyword(s):

Mosaic Virus ◽

Rna Replication ◽

Brome Mosaic Virus ◽

Virus Rna

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Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525616113 ◽

2016 ◽

Vol 113 (8) ◽

pp. 2074-2079 ◽

Cited By ~ 24

Author(s):

Tianyu Jiang ◽

Minhao Liu ◽

Jianping Wu ◽

Yigong Shi

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Virus Protein ◽

B Virus ◽

Iron And Zinc ◽

Important Host ◽

Binding Groove ◽

Α Helix ◽

And Function

HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110–135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

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