Biological Roles and Functional Mechanisms of Arenavirus Z Protein in Viral Replication

Department of Veterinary and Biomedical Sciences, University of Minnesota, Twin Cities, Minnesota, USA.
Journal of Virology (Impact Factor: 4.44). 07/2012; 86(18):9794-801. DOI: 10.1128/JVI.00385-12
Source: PubMed


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.

Download full-text


Available from: Naveen Kumar, Feb 21, 2014
  • Source
    • "At 48 h p.t., we observed fewer GFP-expressing cells (Fig. 5b) and lower levels of Gluc expression (Fig. 5c) in the two-plasmid system than in the four-plasmid system. It is worth noting that in the two-plasmid-based system the Z protein would be expressed by the intracellularly reconstituted virus polymerase complex, and Z is known to inhibit arenavirus RNA replication and transcription (Flatz et al., 2006; Kranzusch & Whelan, 2011; Wang et al., 2012). Despite the observed lower values of reporter gene expressions using the two-plasmid system, we tested the feasibility of using the system to produce rLCMV and rCandid#1 in Vero cells (Fig. 6 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Arenaviruses are important human pathogens with no FDA-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analog ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in the arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, a FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of LCMV directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces to half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live attenuated vaccine Candid#1 strain of the New World arenavirus Junin. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1.
    Full-text · Article · Jan 2013 · Journal of General Virology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Lassa virus (LASV) causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L-a large protein of 2218 amino acid residues-are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP) motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV), which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173) in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1), orthomyxo- (influenza virus PA), and bunyaviruses (La Crosse virus NL1). Although the catalytic residues (D89, E102 and K122) are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever.
    Full-text · Article · Feb 2014 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: Vertebrate innate immunity is characterized by an effective immune surveillance apparatus, evolved to sense foreign structures, such as proteins or nucleic acids of invading microbes. RIG-I-like receptors (RLRs) are key sensors of viral RNA species in the host cell cytoplasm. Activation of RLRs in response to viral RNA triggers an antiviral defense program through the production of hundreds of antiviral effector proteins including cytokines, chemokines, and host restriction factors that directly interfere with distinct steps in the virus life cycle. To avoid premature or abnormal antiviral and proinflammatory responses, which could have harmful consequences for the host, the signaling activities of RLRs and their common adaptor molecule, MAVS, are delicately controlled by cell-intrinsic regulatory mechanisms. Furthermore, viruses have evolved multiple strategies to modulate RLR-MAVS signal transduction to escape from immune surveillance. Here, we summarize recent progress in our understanding of the regulation of RLR signaling through host factors and viral antagonistic proteins.
    No preview · Article · Jun 2014 · Cytokine & Growth Factor Reviews
Show more