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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.  The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes 
PLoS Pathogens  2009;5(5):e1000428.
Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.
Author Summary
The genome of the SARS coronavirus codes for 16 non-structural proteins that are involved in replicating this huge RNA (approximately 29 kilobases). The roles of many of these in replication (and/or transcription) are unknown. We attempt to derive conclusions concerning the possible functions of these proteins from their three-dimensional structures, which we determine by X-ray crystallography. Non-structural protein 3 contains at least seven different functional modules within its 1922-amino-acid polypeptide chain. One of these is the so-called SARS-unique domain, a stretch of about 338 residues that is completely absent from any other coronavirus. It may thus be responsible for the extraordinarily high pathogenicity of the SARS coronavirus, compared to other viruses of this family. We describe here the three-dimensional structure of the SARS-unique domain and show that it consists of two modules with a known fold, the so-called macrodomain. Furthermore, we demonstrate that these domains bind unusual nucleic-acid structures formed by consecutive guanosine nucleotides, where four strands of nucleic acid are forming a superhelix (so-called G-quadruplexes). SUD may be involved in binding to viral or host-cell RNA bearing this peculiar structure and thereby regulate viral replication or fight the immune response of the infected host cell.
doi:10.1371/journal.ppat.1000428
PMCID: PMC2674928  PMID: 19436709

Results 1-2 (2)