View source for User:Wayne Decatur/Flock house virus B2 protein Suppression of RNA Silencing

[[Image:2az0WithPolyview3D.png|left|300px]]<br />


'''3D structure of insect Flock House virus protein B2 complexed to siRNA'''




==Background==

[http://en.wikipedia.org/wiki/RNA_Silencing RNA silencing] is a gene inactivation system in many eukaryotes that relies on tiny RNAs as the targeting molecules. One function of RNA silencing, which is also called [http://en.wikipedia.org/wiki/Post-transcriptional_gene_silencing post-transcriptional gene silencing (PTGS)] or [[RNA Interference|RNA interference]] (RNAi), is to act in surveillance against molecular parasites, such as viruses. Double-stranded RNA triggers the RNA silencing pathway and many viruses use a double-stranded RNA to replicate their genome. Various viruses have developed evasion techniques to circumvent this surveillance system. In one such evasion strategy, Flock House virus protein B2 suppresses the insect's anti-viral RNA-induced silencing response<ref>PMID:12016316</ref>. 
<br>
siRNAs are generally characterized by their short length (21–26 nt), 2 nt, 3′ overhanging ends, and 5′ phosphate groups. The most efficient silencing is obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3' overhang (see [http://www.rockefeller.edu/labheads/tuschl/sirna.html the Tuschl lab's guide] for designing siRNAs)<ref>PMID:11726523</ref><ref>PMID:18280529</ref>.
<br>

A structural study <ref>PMID:16228003</ref> has revealed how Flock House virus protein B2 directly binds double-stranded RNAs that are the size of siRNAs and longer.

==Results==
{{Structure
|PDB= 2az0 |SIZE=400|SCENE=User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/B2view1default/2|CAPTION= insect FHV B2 and siRNA (2az0), resolution 2.60&Aring; (<scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/B2view1default/2'>initial scene</scene>)
|SITE= 
|LIGAND=
|ACTIVITY= 
|GENE= 
|DOMAIN=
|RELATEDENTRY=
|RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2az0 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2az0 OCA], [http://www.ebi.ac.uk/pdbsum/2az0 PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=2az0 RCSB]</span>
}}
The X-ray crystal structure of <scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/Rna_protein/2'>Flock House virus protein B2 complexed with double-stranded RNA</scene> includes both {{User:Wayne Decatur/Template ColorKey Composition lcprotein}} and {{Template:ColorKey Composition RNA}} in the complex.

The <scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/Protein_rna_strands_colored/4'>RNA in the complex is double-stranded and in A-form</scene>.<br>
The synthetic <font color = '#00FF00'>R</font><font color = '#6060ff'>N</font><font color = '#00FF00'>A</font> substrate mimics a small double-stranded [http://en.wikipedia.org/wiki/Small_interfering_RNA siRNAs] that occur in the double-strand RNA-induced RNAi silencing pathway.<br> 
The duplex region here is 18 nts and a biological siRNA has typically a 19 nt duplex and also has additional 2-nt 3' overhangs.<br> 
Here is a schematic illustration of the two strands of the double-stranded RNA in this structure:<br>
<span style="font-weight: bold;font-family: Courier New; font-size: 10pt">
<!-- By setting font to courier, I can use non-proportional font. Whereas by default, rest of Protopedia is proportional -->
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span style="color:#00FF00">5'-pGCAUGGACGCGUCCAUGC-OH-3'<br>&nbsp;&nbsp;
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;||||||||||||||||||<br> 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span style="color:#6060ff">3'-OH-CGUACCUGCGCAGGUACGp-5'</span></span>
<br>

Flock House virus protein B2 (FHV B2) binds the double-stranded RNA as 
<scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/B2view1/8'>a dimer</scene>.<br>
Individual monomers of the B2 dimer are colored <font color = '#cbbe8a'>tan</font> and <font color = 'purple'>purple</font>. <br> 

Each <scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/B2view1second/6'>monomer of FHV B2</scene> is made of <font color = '#ff0080'>three alpha-helices</font>, two long ones and and short one perpendicular to the long ones.

<scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/Fourhelixbundle/6'>The FHV B2 dimer</scene> forms a four-<font color = '#ff0080'>helix</font> bundle. 
<br>
An anti-parallel association of the two B2 monomers forms this four-helix bundle using two helices from each monomer. The third helix in each monomer caps the ends of the bundle. <nowiki>[</nowiki>Note: this view generates a substantial surface which may take half a minute to calculate.<nowiki>]</nowiki><br>

<scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/Proline12/3'>The four-helix bundle of the FHV B2 dimer has a unique proline acting to stabilize it</scene>.
<br>
Unlike canonical left-handed antiparallel four-helix bundles (such as [[1ax8]]) which have a interhelical angle of about 20°, the monomers of B2 are almost linear relative each other.  A '''<font color = 'cyan'>proline (P12)</font>''' kinks helix 1 of the monomer and the unusual feature stabilizes the non-canonical four-helix bundle. Underscoring the unusual nature of this proline is that it is found in a position highly unfavorable based on typical four-helix bundles<ref>PMID:2031185</ref> and that it causes programs for predicting secondary structure to fail to predict the observed helix 1<ref>PMID:8743703</ref>.

<scene name='User:Wayne_Decatur/Flock_house_virus_B2_protein_Suppression_of_RNA_Silencing/B2viewinterface/29'>B2 contacts the phosphate backbone and ribose sugars  to bind the RNA in a sequence independent manner along the whole length of the protein span</scene>.
<br>
Although there is some interaction with the [http://en.wikipedia.org/wiki/RNA deep and narrow major groove], the majority of the interaction by B2 with RNA is with the shallow and wide minor groove.<ref>PMID:8743703</ref>. 
*B2 makes major groove contacts using '''<font color = 'darkorange'>two basic residues (K47 and R54)</font>''' to contact the phosphate backbone. 
*Continuing out from the major groove contacts, B2 makes minor groove contacts using '''<font color = 'aqua'>residues 40, 44, 55, and 58</font>'''.
*Finally, the contacts bridge the minor groove and B2 makes contacts in the minor groove on the opposite strand using '''<font color = 'red'>residues 33, 36, 37, and 62</font>''', which interact with both the phosphates and 2'-hydroxyl.

==Conclusions==
FHV B2 functions as a dimer to bind to one face of double-stranded RNA in a sequence-independent manner. 

Though the four-helix bundle is a common motif (see [[1ax8]],[[1a7m]],[[1c03]],[[1urq]], [[2vrz]] and [[:Category:Four-helix_bundle|other four-helix bundle structures]], for examples)<ref>PMID: 7649401</ref>, recognition of the double-stranded RNA by the B2 four-helix bundle revealed novel mode of RNA binding. Most likely, the B2 protein of Nodamura virus adopts a similar structure as almost all the RNA-contacting residues and Proline 12 are conserved between the two proteins despite marginal sequence similarity <ref>PMID: 11457991</ref>.

The solved structure shows how B2 could directly bind to siRNAs and block use of these intermediates in a defense response to viral attack. Additionally, the structure data and biochemical characterization of B2 binding is consistent with double-stranded RNA longer than 18 bps binding multiple B2 dimers. Such a protective coating of vial double-stranded RNA would inhibit processing by Dicer in the host cell.

In conclusion, the biochemical and structural characterization suggests B2 could act at two stages of RNA silencing. B2 can inhibit both cleavage of dsRNA by Dicer as well as siRNA incorporation into the RNA-induced silencing complex and thus aid viral infection in two ways.

==About this Structure==
2AZ0 is a [[Protein complex]] structure of sequences from [http://en.wikipedia.org/wiki/Nodaviridae Flock House Virus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2az0 OCA]. The molecular weight of FHV B2 protein chain designated 'a' seen in the solved structure is 7.7 kDa (70 residues visible).<!--Calculated using http://www.expasy.org/tools/protparam.html--> The molecular weight of the FHV B2 protein chain designated 'b' seen in the solved structure is 7.8 kDa (71 residues visible). Total size of the FHV B2-RNA complex seen in the structure is 28 kDa. Biological FHV B2 is 106 amino acids and recombinant FHV B2 used to generate the crystals was residues 1-73.

==Reference for the Structure==
Dual modes of RNA-silencing suppression by Flock House virus protein B2., Chao JA, Lee JH, Chapados BR, Debler EW, Schneemann A, Williamson JR, Nat Struct Mol Biol. 2005 Nov;12(11):952-7. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16228003 16228003]

==Related Structures and Topics==
*[[User:Wayne Decatur/Suppression of RNA Silencing by Viruses|Suppression of RNA Silencing by Viruses]]
*[[User:Wayne Decatur/Plant Viral Protein p19 Suppression of RNA Silencing|Plant Viral Protein p19 Suppression of RNA Silencing]]
*[[User:Wayne Decatur/Tomato aspermy virus protein 2b Suppression of RNA Silencing|Tomato aspermy virus protein 2b Suppression of RNA Silencing]]
*[[2az0]] Flock house virus B2 protein bound to double-stranded RNA (dsRNA)
*[[2b9z]] Flock house virus B2 protein solution structure
*[[2zi0]] Tomato aspermy virus protein 2b bound to siRNA
*[[1rpu]] Carnation italian ringspot virus p19 bound to siRNA
*[[1r9f]] Tomato bushy stunt virus p19 bound to siRNA

==Notes and Literature References== 
<references/>
==Additional Literature and Resources==
<ref group="xtra">PMID:16419274</ref>
<references group="xtra"/> 
*The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition., Lingel A, Simon B, Izaurralde E, Sattler M. EMBO Rep. 2005 Dec;6(12):1149-55. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16270100 16270100]<br>
*[http://www-unix.oit.umass.edu/~wdecatur/p19tutorial/ Tour of p19 bound to an siRNA] by Wayne Decatur, in an exploration-friendly interface that is adapted from Eric Martz's [http://molvis.sdsc.edu/fgij/index.htm FirstGlance in Jmol]<br>
*Structural basis for RNA-silencing suppression by Tomato aspermy virus protein 2b., Chen HY, Yang J, Lin C, Yuan YA, EMBO Rep. 2008 Aug;9(8):754-60. Epub 2008 Jul 4.PMID:[http://www.ncbi.nlm.nih.gov/pubmed/18600235 18600235]<br>
*Recognition of small interfering RNA by a viral suppressor of RNA silencing., Ye K, Malinina L, Patel D J, Nature 2003 426(6968):874-878. Epub 2003 Dec 3. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14661029 14661029]<br>
*Sizing up small RNAs., Jabri E, Nature Structural & Molecular Biology 2004 11: 112. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14749769 14749769 ]<br>
*Crystal structure of p19--a universal suppressor of RNA silencing., Baulcombe DC, Molnar A, Trends Biochem Sci. 2004 29(6):279-281. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15276178 15276178]<br>
*Plant viral suppressors of RNA silencing., Roth BM, Pruss GJ, Vance VB, Virus Res. 2004 Jun 1;102(1):97-108. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15068885 15068885]<br>
*Novel modes of protein-RNA recognition in the RNAi pathway. Lingel A, Sattler M, Curr Opin Struct Biol. 2005. 15(1):107-115. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15718141  15718141]<br>
*[http://www.nature.com/focus/rnai/ Nature Reviews RNAi collection]<br>
*The Tombusvirus-encoded P19: from irrelevance to elegance.,Scholthof HB, Nature Reviews Microbiology. 22006 May;4(5):405-11. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16518419 16518419]

[[Category: RNA Silencing]]
[[Category: post-transcriptional gene silencing (PTGS)]]
[[Category: Virus]]
[[Category: Insect Virus]]
[[Category: double-stranded RNA (dsRNA)]]
[[Category: siRNA]]
[[Category: Small Interfering RNA]]
[[Category: Flock house virus]]
[[Category: Single protein]]
[[Category: Chao, J A.]]
[[Category: Chapados, B R.]]
[[Category: Debler, E W.]]
[[Category: Lee, J H.]]
[[Category: Schneemann, A.]]
[[Category: Williamson, J R.]]
[[Category: Four-helix bundle]]
[[Category: Protein-rna complex]]
[[Category: Protein complex]]
[[Category: Rna double helix]]
[[Category: A-form helix]]
[[Category: Rna length recognition]]
[[Category: Rnai]]
[[Category: Nucleus]]
[[Category: Rnai suppression]]
[[Category: Suppressor of rna silencing]]