Complexes assembled from TMV-derived spherical particles and entire virions of heterogeneous nature.

Previously, we described some structural features of spherical particles (SPs) generated by thermal remodelling of the tobacco mosaic virus. The SPs represent a universal platform that could bind various proteins. Here, we report that entire isometric virions of heterogeneous nature bind non-specifically to the SPs. Formaldehyde (FA) was used for covalent binding of a virus to the SPs surface for stabilizing the SP-virus complexes. Transmission and high resolution scanning electron microscopy showed that the SPs surface was covered with virus particles. The architecture of SP-virion complexes was examined by immunologic methods. Mean diameters of SPs and SP-human enterovirus C and SP-cauliflower mosaic virus (CaMV) compositions were determined by nanoparticle tracking analysis (NTA) in liquid. Significantly, neither free SPs nor individual virions were detected by NTA in either FA-crosslinked or FA-untreated compositions. Entirely, all virions were bound to the SPs surface and the SP sites within the SP-CaMV complexes were inaccessible for anti-SP antibodies. Likewise, the SPs immunogenicity within the FA-treated SPs-CaMV compositions was negligible. Apparently, the SP antigenic sites were hidden and masked by virions within the compositions. Previously, we reported that the SPs exhibited adjuvant activity when foreign proteins/epitopes were mixed with or crosslinked to SPs. We found that immunogenicity of entire CaMV crosslinked to SP was rather low which could be due to the above-mentioned masking of the SPs booster. Contrastingly, immunogenicity of the FA-untreated compositions increased significantly, presumably, due to partial release of virions and unmasking of some SPs-buster sites after animals immunization.

RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase in human cells reveals requirements for de novo initiation and protein-protein interaction.

Brome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizes de novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strong cis preference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5'-GUAAA-3') identical to those from the 5' sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a.

Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded proteins, 1a and 2a. The amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs. In this study, we examined the enzymatic activities of BMV 1a expressed in yeast, where the protein is fully functional in RNA replication. 1a methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-adenosylmethionine (AdoMet) as the methyl donor. Product analysis by nuclear magnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7. Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, and was accompanied by transfer of (3)H-methyl from AdoMet to the covalent 1a-guanylate complex. The covalent complex could be immunoprecipitated by 1a antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further methyltransferase reactions. Mutation of conserved amino acids in the N-terminal half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels. Covalent 1a-guanylate complex formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts. These results show that BMV 1a has activities similar to those of alphavirus nsP1, demonstrating conservation of these putative capping functions across a wide span of sequence divergence within the alphavirus-like superfamily. Conservation of this unusual combination of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.