LASV NP N Domian: Cap Binding
The N domain of the LASV NP is characterized by residues 7-338. This domain is crucial for viral transcription to take place. The domain forms a novel structure and this structure has been implicated in the binding of the host’s 5’ RNA m7GpppN cap. Arenaviruses, as well negative RNA strand viruses such as orthomyxoviruse (influenza) and bunyaviruses lack the ability to cap their own RNA transcripts (Fetcher & Brownlee 2005). However they have evolved mechanisms that allow them to ‘snatch’ the 5’ cap off host RNA transcripts allowing viral replication to take place. The LASV NP N domain is crucial to this process as it acts as the 5’ cap binding pocket, allowing endonucleases to cleave the rest of the RNA. In the case of Lassa virus the N domain of LV protein L contains the endonucleases activity required to cleave the host RNA 5’ cap from the rest of the transcript (Lelke, Brunotte, Busch, & Günther 2009). The 5’ cap remains and can be used to initiate transcription of the viral genome by acting as a primer.
LASV NP N Domain Structure:
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| Figure 1.a: The cartoon structure of the LASV NP N domain (Residues 7-338) |
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| Figure 1.b: An image depicting the surface structure of the protein N domain including the proposed cap-binding site in a deep cavity.(Qi et al 2010) were able to co crystallise the LASV NP along with dTTP bound within the cavity of the N Domain) |
Structure of the Cap Binding region:
The dTTP molecule is coordinated within the cavity through interactions with a number of residues. The conserved residues K309, R300, R323 and K253 form salt bonds with the a, b & g phosphates of the triphosphate group of the dTTP (see below).
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| Figure 2.a: The image depicts the N domain 5' mRNA cap binding pocket. The surface in red shows the residues involved in the coordination of the triphosphate group of the substrate (dTTP in this case). |
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| Figure 2.b: The 2nd image focuses on the 4 residues involved in the coordination of the triphosphates of the dTTP. Salt bridges are shown between the triphosphate and the residues K309, R300, R323 and K253. |
Deep within the cavity a hydrophobic pocket of residues F167, W164, L172, M54, L120, L239 and I241 holds in place the thymidine of dTTP (see below).
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| Figure 3.a: The image shows the cap binding domain as in figure 2.a. However also highlighted in yellow are the 7 residues that form the hydrophobic pocket in which sits the aromatic group of the ribonucleotide base. |
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| Figure 3.b: The image focuses in on the 7 key residues that form the hydrophobic pocket: F167, W164, L172, M54, L120, L239 and I241. ----------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
A comparison to known 5'cap binding proteins:
Although not identical this structure shares many common features to other known cap binding proteins such as eukaryote initiation factor 4E. In eukaryote cells this protein is known to bind the 5' mRNA cap to initiate translation. The image below shows eIF4e bound to m7GTP:
Although not identical this structure shares many common features to other known cap binding proteins such as eukaryote initiation factor 4E. In eukaryote cells this protein is known to bind the 5' mRNA cap to initiate translation. The image below shows eIF4e bound to m7GTP:
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| Figure 4. Image showing the mRNA cap binding region of eIf4e (PDB ID 1L8B). Note the similarity to the cap binding region of LASV NP N domain. The aromatic ring of the cap is held in a hydrophobic pocket (W102 and W56). The negative phosphate groups are coordinated by the positively charged K162 and R157 residues. Image courtesy of Fechter & Brownlee, recognition of mRNA cap strucutres by viral and cellular proteins, Journal of General Virology 2005 Vol 86 pg 1242 ---------------------------------------------------------------------------------------------------------------------------------------------------------- |
A second hydrophobic region within the cavity is believed to be the binding site for the 2nd base of the 5’ Cap (m7GpppN). Residues Y319, Y209, Y213, L265 as well as E266 are responsible for this second binding region. The entrance to this cavity has been measured to be 9 Å at it’s narrowest point which ensures the cavity is able to accommodate ss mRNA.
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Figure 5.a: An image showing the 3rd region (orange) involved in the binding of the 5' mRNA cap. The residues involved are shown below |
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| Figure 5.b: An image labeling the 5 residues responsible for the formation of a second hydrophobic region in the cap binding domain. This second hydrophobic region ensures favorable interactions with the second base of the 5' mRNA cap. The residues shown are Y319, Y209, Y213, L265 and E266. |
Alanine scanning mutagenesis studies show the key residues required in cap binding and viral transcription initiation (see methods) K253A and E266A mutants showed no RNA transcriptional activity whilst Y319A, F176A, W164A, K309A and R323A mutants were found to reduce RNA transcriptional activity. R300A produced only a very slight fall in transcriptional activity. Mutations at residues W12A and Y209A had no effect on activity.
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| Results showing the residues involved in cap binding and their roles in viral transcription. Alanine mutations of residues K253 and E266 clearly prevent transcription from taking place suggesting these two residues are fundamental to mRNA cap binding. Other mutations of key residues appear to effect the efficiency of transcription. Data courtesy of Qi et al 2010 |