Functional Analysis Of Smaller Subunit Of Turnip Crinkle Virus RdRp P28in Superinfection Exclusion

Superinfection exclusion (SE) or Homologous interference is a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus. SE has been described for various viruses, including important vertebrte, invertebrte, and plant pathogens. Indeed, SE, which is commonly referred to as "cross-protection", has also been used as a protective measure by purposely infecting plants with mild isolates of a virus to reduce infection and losses due to more severe isolates. Since its first report in1929, cross-protection has been explained in various ways, but unfortunately, its molecular mechanism is still unclear. In this report, I adopted the Turnip crinkle virus (TCV)-Arabidopsis system as a model to study cross-protection. TCV is a small icosahedral virus with a monopartite, positive sense RNA genome of4,054nucleotides (nt) that belongs to the Carmoviruss genus, in the Tombusviridae family and has an extensive host range including Arabidopsis and N. benthamiana.To unravel the molecular mechanism of cross-protection, a series of TCV variants were designed by inserting21nt long oligo nucleotides of different sequences immediately after the CP open reading frame. By inoculating WT and dcl2/dcl4Arabidopsis plants with nine TCV variants, I found that one variant excluded other TCV derivatives during co-infection and showed that plant antiviral gene silencing does not play an important role in TCV cross-protection. Then super-inoculation experiments were conducted between TCV, CCFV(Cardamine chlorotic fleck virus) and CarMV (Carnation mottle virus), As expected from previous studies, I found that cross-protection only occurred closely related viruses. To determine whether exclusion was conferred by an individual TCV protein, I assessed the potential of each TCV protein to repress TCV replication by agrobacterium infiltrations. The results revealed that TCV-encoded P28, which is required for replication, exerts a highly specific and potent repression of TCV replication. By confocal microscopy observations, I was able to demonstrate that P28repressed TCV replication and formed multi-molecular aggregates as large as cell nuclei. Strikingly, the repression of TCV replication by P28did not abolish the translation of P28from the TCV genome. Then I adapted two different promoters for transcription of the mRNA of P28-GFP and P28-Mcherry fusion proteins. The results revealed that P28was able to interfere with P28translation from another source to inhibit TCV replication. Furthermore, semi-denatuirng gel electrophoresis results indicated that P28formed large, SDS-resistant aggregates during TCV infection. These results prompted the hypothesis in which P28plays opposite roles at different stages of the TCV multiplication cycle. First, P28functions in replication of the "protecting" virus early in infection and second, later in infection P28forms large nonfunctional aggregates that sequester P28encoded by subsequently infecting TCV to prevent replication of the second virus. In general, the first portion of my thesis succeeded in unraveling a mechanism for Cross-protection between TCV variants and demonstrated that P28plays an important role by forming large aggregates that inhibit subsequent invasion of different virus strains.Beet black scorch virus (BBSV) is a small single-stranded, positive-sense RNA plant virus belonging to the genus Necrovirus, in the Tombusviridae family. The28nm icosahedral particle encapsidate the3,644nucleotide (nt) monopartite genomic RNA. In a previous study, we identified a motif4KRNKGGKKSR13rich in K and R residues, in the N terminus of the BBSV coat protein. This motif mediates nuclear localization of the CP, and our experiments lead to a model that the K/R-rich motif has an important role in BBSV long distance movement.To further assess the functions of the basic amino acid residues in this motif, a series of mutants were generated by either replacing one or more basic amino acids with alanines (A) or deleting the complete motif. The resulting mutants were exhaustively examined for their replication competence, particle assembly, and systemic spread. First, replication of the mutants was tested in protoplasts, but none of the mutants compromised BBSV RNA replication appreciably. However, the mutants showed varying degrees of assembly defects and stability of BBSV virions, as determined by virus purification and RNase protection assays. These experiments demonstrated that all basic residues within the N-terminal region of the BBSV CP are required for ensure proper assembly of BBSV virions. Furthermore, I was able to determine that4KR5are the two most critical residues required for RNA-CP interactions and virion assembly. These results were further confirmed in vitro by assessing RNA-binding activities of the mutated CPs. I also demonstrated the indispensability of assembled virions for BBSV systemic infections in N. benthamiana by showing that defects in virion assembly/stability caused by the various CP mutants correlated with abundance of virus RNAs in systemically infected leaves. In summary, the N-terminal basic domain of BBSV CP is essential for nuclear localization, efficient CP-RNA interactions and virion stability, and intact virions are required for viral systemic spread.