Juanita McLachlan

The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.

Two Tomato bushy stunt virus (TBSV) proteins, P19 and P22, have been found to interact with the Nicotiana benthamiana host proteins Hin19 and HFi22 in yeast two,hybrid assays. To determine functional roles of these interacting host proteins, viral induced gene silencing (VIGS) was employed to knock,down their expression. TBSV has been demonstrated to activate a virus,specific antiviral response pathway in N. benthamiana. To characterize this pathway, the antiviral RNAi induced silencing complex (RISC) was isolated from TBSV-infected plants. Additionally, putative RISC-associated proteins were identified in silico and suggested roles for these have been identified through literature and database searches. A further aim was the identification of proteins that coimmunoprecipitate with the TBSV-induced RISC following RISC isolation.\n\nA primary aim of this investigation was to identify functional roles for host proteins that interact with the two TBSV 3-terminal encoded proteins, P22 and P19. Each of these has functional roles in viral movement and pathogenicity. In yeast two-hybrid assays, P22 has been shown to interact with HFi22 while P19 interacts with Hin19. VIGS was utilized in attempts to silence the expression of these two host proteins in order to determine their functional roles.\n\nVIGS-mediated suppression of the TBSV-interacting proteins Hin19 and HFi22 has not been accomplished. Despite multiple attempts and multiple approaches, these proteins have not been amenable to silencing. In light of this finding, it is proposed that rather than utilizing VIGS to down-regulate protein levels for Hin19 and HFi22, other approaches should be utilized.\n\nTo characterize the TBSV-mediated RNAi pathway, functionally active antiviral RISC was purified from TBSV-infected N. benthamiana plants using ion-exchange chromatography. This RISC was found to be active only in the degradation of TBSV transcripts, indicating the specificity expected from a programmed RISC.\n\nCharacterization and identification of proteins that copurify with RISC has not yet been accomplished, though in silico analysis has yielded over 150 putative RISC-associated proteins. Of these, a subset has been identified as highly likely candidates based upon function and/or homology to RISC-associated proteins in non-plant organisms, and a model for the TBSV-induced antiviral pathway has been proposed.