NS5B: Difference between revisions

Line 11:

The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.

Empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA.

Empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/3'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/10'>fingers domain</scene>. This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA.

[http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html This image] is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.

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@@ Line 11: / Line 11: @@
 The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.
-Empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA.
+Empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/3'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/10'>fingers domain</scene>. This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA.
 [http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html This image] is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.