The 5′ region of Tacaribe virus L RNA encodes a protein with a potential metal binding domain

Macleod

et al. 2011

The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site-directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50, and Y57, located around the zinc coordination site I, play a critical role in the Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV) self-associates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated with its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homo-oligomerization is not a requirement for Z-L interaction or Z-mediated polymerase activity inhibition.

mentioning

confidence: 99%

Molecular Determinants of Arenavirus Z Protein Homo-Oligomerization and L Polymerase Binding

Loureiro

Macleod

et al. 2011

“…Using this system, we also demonstrated that Z is a powerful inhibitor of RNA replication and transcription (21). Lymphocytic choriomeningitis virus (LCMV) Z protein exhibits a similar inhibitory effect when transcription and replication are analyzed in the homologous (LCMV) minigenome system (6).Arenavirus Z proteins contain a conserved 37-amino-acid (aa) RING domain that comprises about 40% of their amino acid residues (9,16,27,29). The RING domain binds two zinc ions in a cross-brace arrangement and is involved in proteinprotein interactions (2).…”

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confidence: 99%

“…TV, like all arenaviruses, is an enveloped virus containing two single-stranded RNA segments called S and L. The S RNA contains two genes, one encoding the nucleoprotein (N; 64 kDa) and one encoding the glycoprotein precursor (55 kDa) (10), while the L RNA encodes the RNA-dependent RNA polymerase (L; 240 kDa) (15) and a small protein with a RING finger motif (Z; 11 kDa) (16). In both S and L RNAs, the genes are arranged in opposite orientation and are separated by noncoding sequences that have the potential to form stable secondary structures (11).…”

mentioning

confidence: 99%

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Tacaribe Virus Z Protein Interacts with the L Polymerase Protein To Inhibit Viral RNA Synthesis

Jácamo

López

et al. 2003

Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. The TV genome encodes four proteins, the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a small RING finger protein (Z). Using a reverse genetic system, we recently demonstrated that TV N and L are sufficient to drive transcription and full-cycle RNA replication mediated by TV-like RNAs and that Z is a powerful inhibitor of these processes (N. López, R. Jácamo, and M. T. Franze-Fernández, J. Virol. 65:12241-12251, 2001). In the present study we investigated whether Z might interact with either of the proteins, N and L, required for RNA synthesis. To that end, we used coimmunoprecipitation with monospecific antibodies against the viral proteins and coimmunoprecipitation with serum against glutathione S-transferase (GST) and binding to glutathioneSepharose beads when Z was expressed as a fusion protein with GST. We demonstrated that Z interacted with L but not with N and that Z inhibitory activity was dependent on its ability to bind to L. We also evaluated the contribution of different Z regions to its binding ability and functional activity. We found that integrity of the RING structure is essential for Z binding to L and for Z inhibitory activity. Mutants with deletions at the N and C termini of Z showed that amino acids within the C-terminal region and immediately adjacent to the RING domain N terminus contribute to efficient Z-L interaction and are required for inhibitory activity. The data presented here provide the first evidence of an interaction between Z and L, suggesting that Z interferes with viral RNA synthesis by direct interaction with L. In addition, coimmunoprecipitation studies revealed a previously unreported interaction between N and L.Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. Within this group the viruses form three phylogenetically distinct clades, one of which includes TV together with the four known South American pathogens that produce severe hemorrhagic disease (Junin, Machupo, Guanarito, and Sabia viruses) (5). TV, however, does not seem to be a human pathogen.TV, like all arenaviruses, is an enveloped virus containing two single-stranded RNA segments called S and L. The S RNA contains two genes, one encoding the nucleoprotein (N; 64 kDa) and one encoding the glycoprotein precursor (55 kDa) (10), while the L RNA encodes the RNA-dependent RNA polymerase (L; 240 kDa) (15) and a small protein with a RING finger motif (Z; 11 kDa) (16). In both S and L RNAs, the genes are arranged in opposite orientation and are separated by noncoding sequences that have the potential to form stable secondary structures (11). The 5Ј regions of arenavirus genomes and antigenomes, though positively stranded, are not translated directly into proteins. Rather, genomes and antigenomes are found only as nucleocapsids tightly bound to N, and the coding sequences are expressed from mRNAs transcribed from the 3Ј regions of the genomes or antigenomes (1,11,17,19,26). These mRNAs contai...

“…The L RNA encodes the RNA-dependent RNA polymerase (RdRp) (L protein [240 kDa]) (16) and a small protein with a RING finger motif, called Z (11 kDa) (17). In both S and L RNAs, the genes are arranged in opposite orientations and are separated by noncoding sequences that have the potential to form stable secondary structures (14).…”

mentioning

confidence: 99%

Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding

López

Casabona

et al. 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 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.Tacaribe virus (TacV) is the prototype of the New World group of arenaviruses. Within this group, the viruses form three phylogenetically distinct clades, one of which includes TacV together with the known South American pathogens that produce severe hemorrhagic disease: the Junin, Machupo, Guanarito, and Sabia viruses and the recently described Chapare virus (4, 7, 11). TacV, however, seems not to be a human pathogen.TacV is an enveloped virus containing two single-stranded RNA segments called S and L. The S RNA contains two genes encoding respectively the nucleoprotein (N [64 kDa]) and the glycoprotein precursor (55 kDa) of the surface glycoproteins (13). The L RNA encodes the RNA-dependent RNA polymerase (RdRp) (L protein [240 kDa]) (16) and a small protein with a RING finger motif, called Z (11 kDa) (17). In both S and L RNAs, the genes are arranged in opposite orientations and are separated by noncoding sequences that have the potential to form stable secondary structures (14). S and L genomes and antigenomes are found only as nucleocapsids tightly bound to N protein, and the coding sequences are expressed from mRNAs transcribed from the 3Ј region of the genomes and antigenomes (1,14,20,27). These mRNAs end within the intergenic region and contain short stretches of nontemplated nucleotides at their 5Ј ends, which are capped (20,21).Using a reconstituted transcription and replication system based on plasmid-supplied TacV RNAs and proteins, we demonstrated that L and N are the only viral proteins required for full-cycle RNA replication and transcription (22), for a faithful initiation of both processes, and for correct termination of mRNA transcription (21). Using this system, we also demonstrated that TacV Z protein is a potent inhibitor of both viral RNA replication and transcription (22). Lymphocytic choriomeningitis virus (LCMV) Z protein e...