Skip to main content
Article thumbnail
Location of Repository

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

By Ling Liu, William M. Westler, Johan A. den Boon, Xiaofeng Wang, Arturo Diaz, H. Adam Steinberg and Paul Ahlquist

Abstract

Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action

Topics: Research Article
Publisher: Public Library of Science
OAI identifier: oai:pubmedcentral.nih.gov:2654722
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles

    Citations

    1. (1999). A
    2. (1999). A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo.
    3. (2002). A positivestrand RNA virus replication complex parallels form and function of retrovirus capsids.
    4. (2004). Alternate, virusinduced membrane rearrangements support positive-strand RNA virus genome replication.
    5. (2003). An alternate pathway for recruiting template RNA to the brome mosaic virus RNA replication complex.
    6. (2002). An aminoterminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A.
    7. (1997). Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.
    8. (1990). Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis.
    9. (2004). Automated NMR structure calculation with CYANA.
    10. (2005). Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates.
    11. (1996). Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis.
    12. (2000). Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a.
    13. (2001). Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 59 proximal RNA2 signal.
    14. (1998). Brome mosaic virus RNA replication protein 1a dramatically increases in vivo stability but not translation of viral genomic RNA3.
    15. (1999). Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum.
    16. (1992). Bromovirus RNA replication and transcription.
    17. (1997). cis-Acting Signals in Bromovirus RNA Replication and Gene Expression: Networking with Viral Proteins and Host Factors. Seminars in
    18. (1991). Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae.
    19. (2007). Evaluating protein structures determined by structural genomics consortia.
    20. (2006). Evidence for functional protein interactions required for poliovirus RNA replication.
    21. (2002). Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion.
    22. (2000). Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping.
    23. (1987). High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cDNA and effects of 59 extensions on transcript infectivity.
    24. (2005). Highresolution iterative frequency identification for NMR as a general strategy for multidimensional data collection.
    25. (2001). Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes.
    26. (1997). In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae.
    27. (2005). Linear analysis of carbon-13 chemical shift differences and its application to the detection and correction of errors in referencing and spin system identifications.
    28. (2002). Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.
    29. (1984). Multicomponent RNA plant virus infection derived from cloned viral cDNA.
    30. (2006). NMR of membrane proteins in solution.
    31. (2006). NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus.
    32. (1984). Nucleotide sequence of the brome mosaic virus genome and its implications for viral replication.
    33. (2006). Parallels among positive-strand RNA viruses, reversetranscribing viruses and double-stranded RNA viruses.
    34. (2005). Probabilistic Identification of Spin Systems and their Assignments including Coil-Helix Amphipathic Helix Roles
    35. (2005). Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements.
    36. (2002). Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.
    37. (2002). Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS.
    38. (1999). Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.
    39. (1993). RNA-dependent replication, transcription, and persistence of brome mosaic virus RNA replicons in S.
    40. (1999). RNAcontrolled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein.
    41. (2007). Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication.
    42. (1999). Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.
    43. (1999). Sequence and structure-based prediction of eukaryotic protein phosphorylation sites.
    44. (1979). Solid phase synthesis without repetitive acidolysis. Preparation of leucyl-alanylglycyl-valine using 9-fluorenylmethyloxycarbonylamino acids.
    45. (2007). Solution NMR of membrane proteins in bilayer mimics: small is beautiful, but sometimes bigger is better.
    46. (2003). Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus.
    47. (2001). The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein.
    48. (2007). Threedimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.
    49. (2000). Virus Taxonomy.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.