Genetic and Biochemical Evidence for an Oligomeric Structure of the Functional L Polymerase of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus (original) (raw)
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Virology, 1989
The complete RNA sequence of the L protein gene of lymphocytic choriomeningitis virus (LCMV) is presented. It is the first L protein sequence to be obtained for the Arenaviridae, a family of single-stranded RNA viruses which includes Lassa fever virus, and the Tacaribe complex viruses such as Pichinde and the Argentine and Bolivian hemorrhagic fever viruses. It is the largest open reading frame on the L RNA spanning 6633 nucleotides and coding for a 2210 amino acid protein with a calculated molecular weight of 254,529. Antipeptide sera identify a gene product encoded on the L RNA: it has a mass of approximately 200,000 Da and is found in virions and ribonucleoprotein complexes from infected cells (M. Singh, F. Fuller-Pace, M. J. Buchmeier, and P. J. Southern, 1987, Virology, 161, 448-456). Mutations mapped to the L gene affect plaque morphology (Kirk et al., 1980), the lethality of a virulent LCMV strain on guinea pigs (Y. Riviere, R. Ahmed, P. J. Southern, M. J. Buchmeier, and M. B...
Journal of General Virology, 1990
The large (L) protein subunit of unsegmented negativestrand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagree-ment with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis. 0000-9327 © 1990 SGM
Journal of Virology, 2009
. In this study, we have mapped the regions and specific amino acid residues within NP involved in its anti-IFN activity. We identified a region spanning residues 382 to 386 as playing a critical role in the IFN-counteracting activity of NP. Alanine substitutions at several positions within this region resulted in NP mutants that lacked the IFN-counteracting activity but retained their functions in virus RNA synthesis and assembly of infectious particles. We used reverse genetics to rescue a recombinant LCMV strain carrying mutation D382A in its NP [rLCMV/ NP*(D382A)]. Compared to wild-type (WT) LCMV, rLCMV/NP*(D382A) exhibited a higher level of attenuation in IFN-competent than IFN-deficient cells. In addition, A549 cells infected with rLCMV/NP*(D382A), but not with WT LCMV, produced IFN and failed to rescue replication of the IFN-sensitive Newcastle disease virus.
Assembly of a functional Machupo virus polymerase complex
Proceedings of the National Academy of Sciences, 2010
Segmented negative-sense viruses of the family Arenaviridae encode a large polymerase (L) protein that contains all of the enzymatic activities required for RNA synthesis. These activities include an RNA-dependent RNA polymerase (RdRP) and an RNA endonuclease that cleaves capped primers from cellular mRNAs to prime transcription. Using purified catalytically active Machupo virus L, we provide a view of the overall architecture of this multifunctional polymerase and reconstitute complex formation with an RNA template in vitro. The L protein contains a central ring domain that is similar in appearance to the RdRP of dsRNA viruses and multiple accessory appendages that may be responsible for 5′ cap formation. RNA template recognition by L requires a sequence-specific motif located at positions 2–5 in the 3′ terminus of the viral genome. Moreover, L-RNA complex formation depends on single-stranded RNA, indicating that inter-termini dsRNA interactions must be partially broken for complex...
Viruses, 2012
Arenaviruses merit significant interest because several family members are etiological agents of severe hemorrhagic fevers, representing a major burden to public health. Currently, there are no FDA-licensed vaccines against arenaviruses and the only available antiviral therapy is limited to the use of ribavirin that is partially effective. Arenavirus nucleoprotein (NP) is found associated with the genomic RNA forming the viral ribonucleoproteins (vRNPs) that together with the polymerase (L) direct viral replication and transcription. Virion formation requires the recruitment of vRNPs into budding sites, a process in which the arenavirus matrix-like protein (Z) plays a major role. Therefore, proper NP-NP and NP-Z interactions are required for the generation of infectious progeny. In this work we demonstrate the role of the amino acid residue D471 in the self-association of lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP). Amino acid substitutions at this position abrogate NP oligomerization, affecting its ability to mediate replication and transcription of a minigenome reporter plasmid. However, its ability to interact with the Z protein, counteract the cellular interferon response and bind to dsRNA analogs was retained. Additionally, we also document the dominant negative effect of
Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
PLoS pathogens, 2017
RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted p...
Journal of General Virology, 2008
Arenaviruses include several causative agents of haemorrhagic fever disease in humans. In addition, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a superb model for the study of virus-host interactions, including the basis of viral persistence and associated diseases. There is little understanding about the molecular mechanisms concerning the regulation and specific role of viral proteins in modulating arenavirus-host cell interactions either associated with an acute or persistent infection, and associated disease. Here, we report the genomic and biological characterization of LCMV strains 'Docile' (persistent) and 'Aggressive' (not persistent) recovered from cloned cDNA via reverse genetics. Our results confirmed that the cloned viruses accurately recreated the in vivo phenotypes associated with the corresponding natural Docile and Aggressive viral isolates. In addition, we provide evidence that the ability of the Docile strain to persist is determined by the nature of both S and L RNA segments. Thus, our findings provide the foundation for studies aimed at gaining a detailed understanding of viral determinants of LCMV persistence in its natural host, which may aid in the development of vaccines to prevent or treat the diseases caused by arenaviruses in humans.
PLOS Pathogens, 2010
Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a 'capsnatching' 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 a/b 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. Citation: Morin B, Coutard B, Lelke M, Ferron F, Kerber R, et al. (2010) The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription. PLoS Pathog 6(9): e1001038.