Geminiviral βC1 orchestrates organellar genomic instability to augment viral infection by hijacking host RecA

Chloroplast is the site for transforming light energy to chemical energy. It also acts as a production unit for a variety of defense-related molecules. These defense moieties are necessary to mount a successful counter defence against pathogens including viruses. Geminiviruses disrupt chloroplast homeostasis as a basic strategy for their successful infection inducing vein-clearing, mosaics and chlorosis in infected plants. Here we show that a geminiviral pathogenicity determinant protein βC1 directly interferes with plastid homeostasis. βC1 was capable of inducing organelle-specific nuclease to degrade plastid genome as well as diverted functions of RecA1 protein, a major player in plastid genome maintenance. βC1 interacted with RecA1 in plants and its homolog in bacteria to reduce the ability of host cells to maintain genomic integrity under stresses. Further, reduction in the coding capacity of plastids severely affected retrograde signalling necessary for viral perception and activation of defense. Induction of chloroplast-specific nuclease by βC1 is similar to phosphate starvation-response in which nucleotides are recycled to augment synthesis of new, potentially viral, DNA. These results indicate the presence of a novel strategy in which a viral protein alters host defence by targeting regulators of chloroplast DNA. We predict that the mechanism identified here might have similarities in other plant-pathogen interactions.

Geminivirus betasatellite-encoded βC1 protein exhibits novel ATP hydrolysis activity that influences its DNA-binding activity and viral pathogenesis

Plant virus satellites are maintained by their associated helper viruses and satellites influence viral pathogenesis. Diseases caused by geminivirus-betasatellite complexes can become epidemics and therefore have become a threat to economically important crops across the world. Here, we identified a novel molecular function of the betasatellite-encoded pathogenicity determinant βC1. The tomato leaf curl Patna betasatellite (ToLCPaB)-encoded βC1 protein was found to exhibit novel ATPase activity in the presence of the divalent metal ion cofactor MgCl 2 . Moreover, ATPase activity was confirmed to be ubiquitously displayed by βC1 proteins encoded by diverse betasatellites. Mutational and sequence analysis revealed conserved lysine/arginine residues at positions 49/50 and 91 of βC1 proteins to be essential for their ATPase activity. Biochemical studies revealed the DNA-binding activity of the βC1 protein was interfered by the binding of ATP to the protein. Mutating arginine 91 of βC1 to alanine reduced its DNA-binding activity. The results of docking studies provided evidence for an overlap of the ATP-binding and DNA-binding regions of βC1 and for the importance of arginine 91 for both ATP-binding and DNA-binding activities. A mutant betasatellite with a specifically βC1-ATPase dominant negative mutation was found to induce symptoms on Nicotiana benthamiana plants similar to those induced by wild-type betasatellite infection. The ATPase function of βC1 was found to be negatively associated with geminivirus-betasatellite DNA accumulation, despite the positive influence of this ATPase function on the accumulation of replication-associated protein (Rep) and βC1 transcripts. Importance Most satellites influence the pathogenesis of their helper viruses. Here we characterized the novel molecular function of βC1, a non-structural, pathogenicity determinant protein encoded by a betasatellite. Here, we demonstrated the display of ATPase activity by this βC1 protein. Additionally, we confirmed the ubiquitous display of ATPase activity by βC1 proteins encoded by diverse betasatellites. The lysine/arginine residues conserved at positions 49 and 91 of βC1 were found to be crucial for its ATPase function. DNA-binding activity of βC1 was found to be reduced in the presence of ATP. Inhibition of ATPase activity of βC1 in the presence of an excess concentration of cold ATP, GTP, CTP or UTP suggested that the purified βC1 can also hydrolyze other cellular NTPs besides ATP in vitro. These results established the importance of the ATPase and DNA-binding activities of the βC1 protein in regulating the geminivirus-betasatellite DNA accumulation in the infected plant cell.

Stability of Begomoviral pathogenicity determinant βC1 is modulated by mutually antagonistic SUMOylation and SIM interactions

To successfully invade new hosts, to break host resistance as well as to move within and between plant cells, viruses and their satellites have evolved a coordinated network of protein interactions. βC1 protein encoded by specific geminiviral satellites acts as a key pathogenicity determinant. βC1 from diverse viruses undergo multiple post-translational modifications (PTMs) such as ubiquitination and phosphorylation. However, the relevance of these and other layers of PTMs in host-geminiviral interactions has not been fully understood. Here we identified the significance of a novel layer of PTMs in Synedrella yellow vein clearing virus (SyYVCV) encoded βC1 protein having well conserved SUMOylation and SUMO-interacting motifs (SIMs). We observed that SyYVCV βC1 undergoes SUMOylation in host plants as a defensive strategy against ubiquitin mediated degradation. On the contrary, SIMs encoded in βC1 mediate degradation of βC1. Both these PTMs are also essential for the function of βC1 protein since SIM and SUMOylation motif mutants failed to promote pathogenicity and viral replication in vivo. In addition, SUMOylation in different motifs of βC1 led to functionally distinct outcomes, regulating the stability and function of the βC1 protein, as well as increased global SUMOylation of host proteins. Our results indicate the presence of a novel mechanism mediating a fine balance between defence and counter-defence in which a SIM site is competitively sought for degradation and as a counter defense, βC1 undergoes SUMOylation to escape from its degradation.Summary StatementβC1 viral protein has evolved counter-defensive strategies to perturb host protein degradation pathways

A PB1-K577E Mutation in H9N2 Influenza Virus Increases Polymerase Activity and Pathogenicity in Mice

H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation.