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1.  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
2.  In Vitro Suppression of K65R Reverse Transcriptase-Mediated Tenofovir- and Adefovir-5′-Diphosphate Resistance Conferred by the Boranophosphonate Derivatives▿  
9-[2-(Boranophosphonomethoxy)ethyl]adenine diphosphate (BH3-PMEApp) and (R)-9-[2-(boranophosphonomethoxy)propyl]adenine diphosphate (BH3-PMPApp), described here, represent the first nucleoside phosphonates modified on their α-phosphates that act as efficient substrates for the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). These analogues were synthesized and evaluated for their in vitro activity against wild-type (WT), K65R, and R72A RTs. BH3-PMEApp and BH3-PMPApp exhibit the same inhibition properties as their nonborane analogues on WT RT. However, K65R RT was found hypersensitive to BH3-PMEApp and as sensitive as WT RT to BH3-PMPApp. Moreover, the presence of the borane group restores incorporation of the analogue by R72A HIV RT, the latter being nearly inactive with regular nucleotides. The BH3-mediated suppression of HIV-1 RT resistance, formerly described with nucleoside 5′-(α-p-borano)-triphosphate analogues, is thus also conserved at the phosphonate level. The present results show that an α-phosphate modification is also possible and interesting for phosphonate analogues, a result that might find application in the search for a means to control HIV RT-mediated drug resistance.
doi:10.1128/AAC.00145-07
PMCID: PMC2043169  PMID: 17620380
3.  Structural and Functional Basis for ADP-Ribose and Poly(ADP-Ribose) Binding by Viral Macro Domains 
Journal of Virology  2006;80(17):8493-8502.
Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona- and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Å resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1"-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.
doi:10.1128/JVI.00713-06
PMCID: PMC1563857  PMID: 16912299

Results 1-3 (3)