Nucleic Acid Affinity Chromatography: Preparation and Characterization of Double-Stranded RNA Agarose

The host antiviral protein kinase R (PKR) has rapidly evolved during primate evolution, likely in response to challenges posed by many different viral antagonists, such as the TRS1 gene of cytomegaloviruses (CMVs). In turn, viral antagonists have adapted to changes in PKR. As a result of this "arms race," modern TRS1 alleles in CMVs may function differently in cells derived from alternative species. We have previously shown that human CMV TRS1 (HuTRS1) blocks the PKR pathway and rescues replication of a vaccinia virus mutant lacking its major PKR antagonist in human cells. We now demonstrate that HuTRS1 does not have these activities in Old World monkey cells. Conversely, the rhesus cytomegalovirus homologue of HuTRS1 (RhTRS1) fulfills these functions in African green monkey cells, but not rhesus or human cells. Both TRS1 proteins bind to double-stranded RNA and, in the cell types in which they can rescue VV⌬E3L replication, they also bind to PKR and prevent phosphorylation of the ␣-subunit of eukaryotic initiation factor 2. However, while HuTRS1 binds to inactive human PKR and prevents its autophosphorylation, RhTRS1 binds to phosphorylated African green monkey PKR. These studies reveal that evolutionary adaptations in this critical host defense protein have altered its binding interface in a way that has resulted in a qualitatively altered mechanism of PKR antagonism by viral TRS1 alleles from different CMVs. These results suggest that PKR antagonism is likely one of the factors that contributes to species specificity of cytomegalovirus replication.

Section: Cells Virus and Infections Human Fibroblasts (Hf)mentioning

confidence: 99%

Species Specificity of Protein Kinase R Antagonism by Cytomegalovirus TRS1 Genes

Child

Brennan

Braggin

et al. 2012

“…Therefore, we analyzed the ability of the E3-⌬SIM mutant to bind to dsRNA. This was assayed by a poly(I:C)-agarose-binding assay as previously described (37). Both the 35 S-labeled in vitro-synthesized WT-E3 and E3-⌬SIM proteins bound to dsRNA-agarose but not to agarose alone (Fig.…”

mentioning

confidence: 99%

Regulation of Vaccinia Virus E3 Protein by Small Ubiquitin-Like Modifier Proteins

González-Santamaría

Campagna

García

et al. 2011

The vaccinia virus (VACV) E3 protein is essential for virulence and has antiapoptotic activity and the ability to impair the host innate immune response. Here we demonstrate that E3 interacts with SUMO1 through a small ubiquitin-like modifier (SUMO)-interacting motif (SIM). SIM integrity is required for maintaining the stability of the viral protein and for the covalent conjugation of E3 to SUMO1 or SUMO2, a modification that has a negative effect on the E3 transcriptional transactivation of the p53-upregulated modulator of apoptosis (PUMA) and APAF-1 genes. We also demonstrate that E3 is ubiquitinated, a modification that does not destabilize the wild-type protein but triggers the degradation of an E3-⌬SIM mutant. This report constitutes the first demonstration of the important roles that both SUMO and ubiquitin play in the regulation of the VACV protein E3.

“…Linearized pGenCAT⌬ DNA was used as the template for in vitro transcription employing a T7 RNA polymerase-based transcription kit (MegaScript-Ambion). The RNA transcript (MG-CAT⌬), which contains the complete TCRV S 5= and 3= UTRs flanking a truncated version of the CAT gene, was purified by Illustra MicroSpin G-50 columns (GE Healthcare Life Sciences) and then immobilized onto agarose beads according to a well-established procedure (31,32). Briefly, 10 g of synthetic RNA was activated by incubation in 5 mM sodium m-periodate (Sigma-Aldrich)-100 mM sodium acetate (pH 5.0) for 1 h at room temperature in the dark.…”

Section: Cells and Virus Bsr (A Clone Of Bhk-21mentioning

confidence: 99%

Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

D'antuono

Loureiro

Foscaldi

et al. 2014

The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCEThe mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be determined. In this report, novel findings are provided on critical interactions between the viral ribonucleoprotein components. We identify several amino acid residues in both the N-terminal and C-terminal domains of TCRV NP that differentially contribute to NP-NP and NP-RNA interactions and analyze their relevance for binding of NP to the L polymerase and for nucleocapsid activity. Our results provide insight into the contribution of NP self-interaction to RNP assembly and activity and reveal the involvement of the NP C-terminal domain in RNA binding. V iruses belonging to the Arenaviridae family are classified into two main groups, Old World (OW) and New World (NW), according to phylogeny, serological properties, and geographical distribution (1). Several members of this family, such as the NW Junin virus (JUNV) and the OW Lassa virus (LASV), produce serious hemorrhagic fever in humans. Tacaribe virus (TCRV), the prototype of NW arenaviruses, is nonpathogenic, closely related to JUNV, and has been used as a model in numerous studies (1-4). Arenaviruses ar...