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1.  Structural Properties of the C Terminus of Vesicular Stomatitis Virus N Protein Dictate N-RNA Complex Assembly, Encapsidation, and RNA Synthesis 
Journal of Virology  2012;86(16):8720-8729.
The vesicular stomatitis virus (VSV) nucleoprotein (N) associates tightly with the viral genomic RNA. This N-RNA complex constitutes the template for the RNA-dependent RNA polymerase L, which engages the nucleocapsid via its phosphoprotein cofactor P. While N and P proteins play important roles in regulating viral gene expression, the molecular basis of this regulation remains incompletely understood. Here we show that mutations in the extreme C terminus of N cause defects in viral gene expression. To determine the underlying cause of such defects, we examined the effects of the mutations separately on encapsidation and RNA synthesis. Expression of N together with P in Escherichia coli results predominantly in the formation of decameric N-RNA rings. In contrast, nucleocapsid complexes containing the substitution NY415A or NK417A were more loosely coiled, as revealed by electron microscopy (EM). In addition, the NEF419/420AA mutant was unable to encapsidate RNA. To further characterize these mutants, we engineered an infectious cDNA clone of VSV and employed N-RNA templates from those viruses to reconstitute RNA synthesis in vitro. The transcription assays revealed specific defects in polymerase utilization of the template that result in overall decreased RNA quantities, including reduced amounts of leader RNA. Passage of the recombinant viruses in cell culture led to the accumulation of compensatory second-site mutations in close proximity to the original mutations, underscoring the critical role of structural features within the C terminus in regulating N function.
doi:10.1128/JVI.00990-12
PMCID: PMC3421751  PMID: 22674999
2.  The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription 
PLoS Pathogens  2010;6(9):e1001038.
Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
Author Summary
The Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
doi:10.1371/journal.ppat.1001038
PMCID: PMC2940758  PMID: 20862324

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