An Aurora B-RPA signaling axis secures chromosome segregation fidelity.

Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

AC4 protein of tomato leaf curl Palampur virus is an RNA silencing suppressor and a pathogenicity determinant.

Plants deploy RNA silencing as a natural defence against invading viruses involving sequence-specific degradation of the viral RNAs. As a counter-defence strategy, viruses encode suppressor proteins that simultaneously target different steps of the silencing machinery. Tomato leaf curl Palampur virus (ToLCPalV) is a bipartite begomovirus in Geminiviridae family. It is responsible for significant reduction in the crop yield and quality. DNA-A of the virus encodes for six proteins whereas DNA-B codes for two proteins. In this study, all viral genes were screened for their role in suppression of green fluorescent protein (GFP) silencing in Nicotiana tabacum cv. Xanthi, employing agrobacterium based co-infiltration assay. The assay identified AC4 as a potential suppressor of RNA silencing. In addition, AC4 expression also suppressed virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in N. benthamiana. Potato virus X (PVX) mediated transient expression of the AC4 in N. benthamiana showed enhanced symptoms that include downward leaf curling, leaf puckering and tissue necrosis. Further, N. benthamiana lines stably expressing AC4 showed severe developmental abnormalities. Mutational analysis suggested that glycine at 2nd position is essential for AC4 pathogenicity. Collectively, these findings demonstrate the role of ToLCPalV AC4 in viral pathogenesis, disease establishment and suppression of gene silencing.

AV2 protein of tomato leaf curl Palampur virus promotes systemic necrosis in Nicotiana benthamiana and interacts with host Catalase2.

Tomato leaf curl Palampur virus (ToLCPalV) is a whitefly-transmitted, bipartite begomovirus. Here, we demonstrated that ectopic expression of AV2 from a Potato virus X (PVX)-based vector accelerated systemic necrosis and reactive oxygen species (ROS) accumulation in Nicotiana benthamiana. Furthermore, 10 amino acids from N-terminal region of AV2 were found to be associated with the systemic necrosis symptom/phenotype. Mutational studies of ToLCPalV infectious clones lacking the AV2 revealed that AV2 is essential for the systemic movement of DNA-A, symptom severity and viral DNA accumulation. In a yeast two-hybrid assay, Catalase2 (Cat2) was found to associate with AV2 protein. Further, silencing of Cat2 resulted in appearance of necrotic lesions on N. benthamiana and these plants were highly susceptible to ToLCPalV infection in comparison to control plants. Infection ToLCPalV on Solanum lycopersicum resulted in downregulation of Cat2 transcripts, followed by accumulation of ROS and stress marker transcripts. The AV2 protein also suppressed virus-induced gene silencing (VIGS) of the Phytoene desaturase (PDS) gene. Our results show that AV2 is essential for the pathogenicity, systemic movement and suppression of gene silencing in the host. Altogether, our findings suggest that interactions between AV2 and Cat2 might play a crucial role in the establishment of ToLCPalV infection.

Molecular characterization of a new begomovirus infecting Mirabilis jalapa in northern India.

Begomoviruses are whitefly-transmitted single-stranded DNA viruses that are responsible for considerable economic losses. A begomovirus, alphasatellite and betasatellite were characterized in a Mirabilis jalapa plant exhibiting severe leaf curling and mottling symptoms. The complete viral genome shared highest sequence identity of 87% with pedilanthus leaf curl virus (AM712436), reported from Pakistan. Additionally, the viral genome was 84% identical to that of chilli leaf curl India virus (KX951415) and 83% identical to that of tobacco curly shoot virus (GU1999584), which were previously reported to infect M. jalapa in India and China, respectively. Based on the ICTV criterion for begomovirus species demarcation (≥91% sequence identity for the complete genome), the virus represents a new species, for which we propose the name Mirabilis leaf curl virus. The alphasatellite and betasatellite sequences were similar to the corresponding sequences of ageratum yellow vein India alphasatellite (KU852743; 99% identity) and tomato leaf curl Patna betasatellite (HQ180394; 86% identity) sequences, respectively. This report describes a new begomovirus-satellite disease complex in M. jalapa.

Identification of host cellular targets of AC4 and AV2 proteins of tomato leaf curl palampur virus and their sub-cellular localization studies.

Tomato leaf curl palampur virus (ToLCPalV) is a bipartite begomovirus with genome organization typical of old world begomoviruses. It infects commercially important crops and weeds in the Asian subcontinent. Apart from other proteins, the DNA-A of the virus encodes AV2 and AC4 proteins of approximately 13.73 and 6.7 kDa, respectively. In case of other begomoviruses, previous studies have shown the role of AV2 and AC4 proteins in virus movement, pathogenesis and suppression of gene silencing. However, the ToLCPalV proteins are significantly variable in comparison to closest relative and hence there is a need to work out their functions. In this study, we identified 9 cellular proteins of tomato that interact with AV2 and AC4 proteins, through yeast two hybrid screening. Upon sequence analysis, these interactors were identified as cysteine protease, katanin p60 ATPase-containing subunit A-like, guanine deaminase, NADH dehydrogenase (ubiquinone) iron-sulfur protein, glyceraldehyde-3-phosphate dehydrogenase B, 60S acidic ribosomal P0 protein, acyl co-A dehydrogenase IBR3, oxygen-evolving enhancer protein 1 and peroxisomal membrane protein 11D. These proteins play a vital role in protein degradation, plant defense response, microtubule severing, photosynthesis and protein synthesis. The two viral proteins, however, did not interact with each other in yeast. AV2 when fused with GFP under the control of cauliflower mosaic virus 35S promoter was localized in nucleus and cytoplasm. On the other hand, AC4-GFP fusion was localized only in cytoplasm. The outcome of present study will help to elucidate the mechanism of viral pathogenesis. Further functional characterization of identified host proteins will provide an insight into their involvement in disease development.