Characterization of the Arenavirus RING Finger Z Protein Regions Required for Z-Mediated Inhibition of Viral RNA Synthesis

ABSTRACT The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an enveloped virus with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides, namely, nucleoprotein; surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Using a reverse genetic system based on the ARM strain of LCMV, we have previously shown that Z has a strong inhibitory activity on LCMV minigenome transcription and RNA replication (T. I. Cornu and J. C. de la Torre, J. Virol. 75:9415-9426, 2001). In the present study, we have identified regions and specific amino acid residues within Z which contribute to its inhibitory activity on RNA synthesis mediated by the LCMV polymerase. Z proteins from different LCMV strains had similar inhibitory activities on the expression of the LCMV ARM minigenome, whereas the Z protein of the genetically more distantly related Tacaribe virus had an approximately 10-fold lower inhibitory activity on ARM minigenome expression. Results from the use of chimera proteins between Z and Xenopus Neuralized, a nonviral RING finger protein, indicated that the structural integrity of the Z RING domain (RD) was required but not sufficient for the inhibitory activity of Z. Serial deletion mutants of the N and C termini of Z showed that the N terminus (residues 1 through 16) and C terminus (residues 79 through 90) do not contribute to the Z inhibitory activity. A highly conserved tryptophan (W) residue located at position 36 in ARM-Z, next to the second conserved cysteine (C) residue of the Z RD, also contributed to the Z inhibitory activity.

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

Journal of Virology ◽

10.1128/jvi.77.19.10383-10393.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10383-10393 ◽

Cited By ~ 59

Author(s):

Rodrigo Jácamo ◽

Nora López ◽

Maximiliano Wilda ◽

María T. Franze-Fernández

Keyword(s):

Inhibitory Activity ◽

Rna Synthesis ◽

Ring Finger ◽

Direct Interaction ◽

Viral Rna ◽

Reverse Genetic System ◽

Small Ring ◽

Protein Z ◽

Glycoprotein Precursor ◽

Tacaribe Virus

ABSTRACT 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 glutathione-Sepharose 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.

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The RING Domain and the L79 Residue of Z Protein Are Involved in both the Rescue of Nucleocapsids and the Incorporation of Glycoproteins into Infectious Chimeric Arenavirus-Like Particles

Journal of Virology ◽

10.1128/jvi.00329-09 ◽

2009 ◽

Vol 83 (14) ◽

pp. 7029-7039 ◽

Cited By ~ 51

Author(s):

Juan Cruz Casabona ◽

Jesica M. Levingston Macleod ◽

Maria Eugenia Loureiro ◽

Guillermo A. Gomez ◽

Nora Lopez

Keyword(s):

Biochemical Analysis ◽

Ring Finger ◽

Ring Structure ◽

Ring Finger Protein ◽

Protein Z ◽

Glycoprotein Complex ◽

Finger Protein ◽

Tacaribe Virus ◽

Z Protein ◽

Negative Sense

ABSTRACT Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome that encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L), and a RING finger protein (Z), which is the driving force of arenavirus budding. We have established a plasmid-based system which allowed the successful packaging of TacV-like nucleocapsids along with Z and GP of JunV into infectious virus-like particles (VLPs). By coexpressing different combinations of the system components, followed by biochemical analysis of the VLPs, the requirements for the assembly of both N and GP into particles were defined. We found that coexpression of N with Z protein in the absence of minigenome and other viral proteins was sufficient to recruit N within lipid-enveloped Z-containing VLPs. In addition, whereas GP was not required for the incorporation of N, coexpression of N substantially enhanced the ratio of GP to Z into VLPs. Disruption of the RING structure or mutation of residue L79 to alanine within Z protein, although it had no effect on Z self-budding, severely impaired VLP infectivity. These mutations drastically altered intracellular Z-N interactions and the incorporation of both N and GP into VLPs. Our results support the conclusion that the interaction between Z and N is required for assembly of both the nucleocapsids and the glycoproteins into infectious arenavirus budding particles.

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RING Finger Z Protein of Lymphocytic Choriomeningitis Virus (LCMV) Inhibits Transcription and RNA Replication of an LCMV S-Segment Minigenome

Journal of Virology ◽

10.1128/jvi.75.19.9415-9426.2001 ◽

2001 ◽

Vol 75 (19) ◽

pp. 9415-9426 ◽

Cited By ~ 97

Author(s):

Tatjana I. Cornu ◽

Juan Carlos de la Torre

Keyword(s):

Ring Finger ◽

Rna Replication ◽

Genetic System ◽

Lymphocytic Choriomeningitis ◽

Lymphocytic Choriomeningitis Virus ◽

Surface Glycoprotein ◽

Reverse Genetic System ◽

Protein Z ◽

Susceptibility To Infection ◽

Z Protein

ABSTRACT Arenaviruses have a bisegmented negative-strand RNA genome whose proteomic capability is limited to only four polypeptides, namely, nucleoprotein (NP), surface glycoprotein (GP) that is proteolytically processed into GP1+GP2, polymerase (L), and a small (11-kDa) RING finger protein (Z). The role of Z during the Lymphocytic choriomeningitis virus (LCMV) life cycle is poorly understood. We investigated the function of Z in virus transcription and replication by using a reverse genetic system for the prototypic arenavirus LCMV. This system involves an LCMV minigenome and the minimal viral trans-acting factors (NP and L), expressed from separated cotransfected plasmids. Cotransfection of the Z cDNA strongly inhibited LCMV minigenome expression. The effect required synthesis of Z protein; its magnitude was dose dependent and occurred with levels of Z protein substantially lower than those observed in LCMV-infected cells. Coexpression of Z did not prevent the encapsidation of plasmid supplied minigenome, but it affected both transcription and RNA replication similarly. Mutations in Z that unfolded its RING finger domain eliminated its inhibitory activity, but RING proteins not related to Z did not affect LCMV minigenome expression. Consistent with the minigenome results, cells transiently expressing Z exhibited decreased susceptibility to infection with LCMV.

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The small RING finger protein Z drives arenavirus budding: Implications for antiviral strategies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2133782100 ◽

2003 ◽

Vol 100 (22) ◽

pp. 12978-12983 ◽

Cited By ~ 233

Author(s):

M. Perez ◽

R. C. Craven ◽

J. C. de la Torre

Keyword(s):

Ring Finger ◽

Small Ring ◽

Ring Finger Protein ◽

Protein Z ◽

Finger Protein

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Cells Expressing the RING Finger Z Protein Are Resistant to Arenavirus Infection

Journal of Virology ◽

10.1128/jvi.78.6.2979-2983.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2979-2983 ◽

Cited By ~ 34

Author(s):

Tatjana I. Cornu ◽

Heinz Feldmann ◽

Juan Carlos de la Torre

Keyword(s):

Recombinant Adenovirus ◽

Ring Finger ◽

Hemorrhagic Fever ◽

Lassa Fever ◽

Lymphocytic Choriomeningitis ◽

Surface Glycoprotein ◽

Protein Z ◽

Virus Transcription ◽

Lassa Fever Virus ◽

Z Protein

ABSTRACT Arenaviruses include Lassa fever virus (LFV) and the South American hemorrhagic fever viruses. These viruses cause severe human disease, and they pose a threat as agents of bioterrorism. Arenaviruses are enveloped viruses with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides: nucleoprotein (NP); surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Our investigators have previously shown that Z has a strong inhibitory activity on RNA synthesis mediated by the polymerase of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). In this report we show that cells transduced with a replication-deficient recombinant adenovirus expressing Z (rAd-Z) are resistant to LCMV and LFV infection. Virus cell entry mediated by LCMV or LFV GP was not affected in rAd-Z-transduced cells, but both virus transcription and replication were strongly and specifically inhibited, which resulted in a dramatic reduction in production of infectious virus. These findings open new avenues for developing antiviral strategies to combat the highly pathogenic human arenaviruses, including LFV.

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