The Role of Arena Virus Protein and Their Molar Ratio (original) (raw)
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Journal of Virology, 2005
The arenavirus L protein has the characteristic sequence motifs conserved among the RNA-dependent RNA polymerase L proteins of negative-strand (NS) RNA viruses. Studies based on the use of reverse-genetics approaches have provided direct experimental evidence of the key role played by the arenavirus L protein in viral-RNA synthesis. Sequence alignment shows six conserved domains among L proteins of NS RNA viruses. The proposed polymerase module of L is located within its domain III, which contains highly conserved amino acids within motifs designated A and C. We have examined the role of these conserved residues in the polymerase activity of the L protein of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), in vivo using a minigenome rescue assay. We show here that the presence of sequence SDD, a characteristic of motif C of segmented NS RNA viruses, as well as the presence of the highly conserved D residue within motif A of L proteins, is strictly required for t...
Uncovering Viral Protein-Protein Interactions and their Role in Arenavirus Life Cycle
Viruses, 2012
The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key proteinprotein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein) and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article.
Arenavirus Quasispecies and Their Biological Implications
Current Topics in Microbiology and Immunology, 2015
The family Arenaviridae currently comprises over 20 viral species, each of them associated with a main rodent species as the natural reservoir and in one case possibly phyllostomid bats. Moreover, recent findings have documented a divergent group of arenaviruses in captive alethinophidian snakes. Human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious materials. Arenaviruses merit interest both as highly tractable experimental model systems to study acute and persistent infections and as clinically important human pathogens including Lassa (LASV) and Junin (JUNV) viruses, the causative agents of Lassa and Argentine hemorrhagic fevers (AHFs), respectively, for which there are no FDA-licensed vaccines, and current therapy is limited to an off-label use of ribavirin (Rib) that has significant limitations. Arenaviruses are enveloped viruses with a bi-segmented negative strand (NS) RNA genome. Each genome segment, L (ca 7.3 kb) and S (ca 3.5 kb), uses an ambisense coding strategy to direct the synthesis of two polypeptides in opposite orientation, separated by a noncoding intergenic region (IGR). The S genomic RNA encodes the virus nucleoprotein (NP) and the precursor (GPC) of the virus surface glycoprotein that mediates virus receptor recognition and cell entry via endocytosis. The L genome RNA encodes the viral RNA-dependent RNA polymerase (RdRp, or L polymerase) and the small (ca 11 kDa) RING finger protein Z that has functions of a bona fide matrix protein including directing virus budding. Arenaviruses were thought to be relatively stable genetically with intra-and interspecies amino acid sequence identities of 90-95 % and 44-63 %, respectively. However, recent evidence has documented extensive arenavirus genetic variability in the field. Moreover, dramatic phenotypic differences have been documented among closely related LCMV isolates. These data provide strong evidence of viral quasispecies
Journal of general …, 1997
The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya-and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS-lymphocytic choriomeningitis and Tacaribe-Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells. Lassa fever virus (LAS), the most dangerous human pathogen among the Arenaviridae, has two single-stranded RNA genomic segments termed large (L) and small (S) (Lukashevich et al., 1984). The LAS S RNA (3.4 kb) encodes the nucleoprotein (NP) and the envelope glycoprotein (GP) in an ambisense coding arrangement (Clegg & Oram, 1985; Auperin et al., 1986). Arenavirus L RNA segments have been described for lymphocytic choriomeningitis (LCM), Tacaribe (TAC) and Pichinde (PIC) viruses (Salvato & Shimomaye, 1989; Iapalucci et al., 1989; D. G. Harnish, S. Zheng & S. Polyak, unpublished results). The L segment (7.2 kb) encodes the viral RNA-dependent RNA polymerase (RdRp or L protein) on the genomecomplementary strand and an 11 kDa ring-finger protein (Z) on the genomic strand. Both of these proteins are involved in regulation of transcription and replication processes and one or both may be implicated in the pathogenic potential of the virus (Salvato, 1993). We report here the sequence of the LAS L gene. LAS L RNA was obtained by immunoprecipitating nucleocapsids from infected cells, extracting them with phenolchloroform, and isolating the L RNA segment on linear 15-30 % sucrose/0-5 % SDS gradients
Biological Roles and Functional Mechanisms of Arenavirus Z Protein in Viral Replication
Journal of Virology, 2012
Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.
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.
Observation of arenavirus nucleoprotein heptamer assembly
FEBS Open Bio, 2021
Arenaviruses are enveloped viruses containing a segmented, negative, and ambisense single-stranded RNA genome wrapped with a nucleoprotein (NP). The NP is the most abundant viral protein in infected cells and plays a critical role in both replication/transcription and virion assembly. The NP associates with RNA to form a ribonucleoprotein (RNP) complex, and this implies self-assembly while the exact structure of this polymer is not yet known. Here, we report a measurement of the full-length Mopeia virus NP by negative stain transmission electron microscopy. We observed RNP complex particles with diameter 15 AE 1 nm as well as symmetric circular heptamers of the same diameter, consistent with previous observations.
Journal of Virology, 2008
Tacaribe virus (TacV) is the prototype of the New World group of arenaviruses. The TacV genome encodes four proteins: the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a RING finger protein (Z). Using a reverse genetics system, we demonstrated that TacV N and L are sufficient to drive transcription and replication mediated by TacV-like RNAs and that Z is a powerful inhibitor of these processes (Lopez et al., J. Virol. 65:12241-12251, 2001). More recently, we provided the first evidence of an interaction between Z and L and showed that Z's inhibitory activity was dependent on its ability to bind to L (Jácamo et al., J. Virol. 77:10383-10393, 2003). In the present study, we mapped the TacV Z-binding sites on the 2,210-amino-acid L polymerase. To that end, we performed deletion analysis and point mutations of L and studied the Z-L interaction by coimmunoprecipitation with specific sera. We found that the C-terminal region of L was not essential for the interaction and identified two noncontiguous regions that were critical for binding: one at the N-terminus of L between residues 156 and 292 and a second one in the polymerase domain (domain III). The importance of domain III in binding was revealed by substitutions in D1188 and H1189 within motif A and in each residue of the conserved SDD sequence (residues 1328, 1329, and 1330) within motif C. Our results showed that of the substituted residues, only H1189 and D1329 appeared to be critically involved in binding Z.