Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

The siRNA sequence and guide strand overhangs are determinants of in vivo duration of silencing.

The use of short interfering RNAs (siRNA) in animals for target validation or as potential therapeutics is hindered by the short physical half-life when delivered as unencapsulated material and in turn the short active half-life of siRNAs in vivo. Here we demonstrate that the character of the two 3'-overhang nucleotides of the guide strand of siRNAs is a determinant of the duration of silencing by siRNAs both in vivo and in tissue culture cells. We demonstrate that deoxyribonucleotides in the guide strand overhang of siRNAs have a negative impact on maintenance of both the in vitro and in vivo activity of siRNAs over time. Overhangs that contain ribonucleotides or 2'-O-methyl modified nucleotides do not demonstrate this same impairment. We also demonstrate that the sequence of an siRNA is a determinant of the duration of silencing of siRNAs directed against the same target even when those siRNAs have equivalent activities in vitro. Our experiments have determined that a measurable duration parameter exists, distinct from both maximum silencing ability and the potency of siRNAs. Our findings provide information on incorporating chemically modified nucleotides into siRNAs for potent, durable therapeutics and also inform on methods used to select siRNAs for therapeutic and research purposes.

Inhibition of transforming growth factor beta signaling reduces pancreatic adenocarcinoma growth and invasiveness.

Transforming growth factor beta (TGFbeta) is a pleiotropic factor that regulates cell proliferation, angiogenesis, metastasis, and immune suppression. Dysregulation of the TGFbeta pathway in tumor cells often leads to resistance to the antiproliferative effects of TGFbeta while supporting other cellular processes that promote tumor invasiveness and growth. In the present study, SD-208, a 2,4-disubstituted pteridine, ATP-competitive inhibitor of the TGFbeta receptor I kinase (TGFbetaRI), was used to inhibit cellular activities and tumor progression of PANC-1, a human pancreatic tumor line. SD-208 blocked TGFbeta-dependent Smad2 phosphorylation and expression of TGFbeta-inducible proteins in cell culture. cDNA microarray analysis and functional gene clustering identified groups of TGFbeta-regulated genes involved in metastasis, angiogenesis, cell proliferation, survival, and apoptosis. These gene responses were inhibited by SD-208. Using a Boyden chamber motility assay, we demonstrated that SD-208 inhibited TGFbeta-stimulated invasion in vitro. An orthotopic xenograft mouse model revealed that SD-208 reduced primary tumor growth and decreased the incidence of metastasis in vivo. Our findings suggest mechanisms through which TGFbeta signaling may promote tumor progression in pancreatic adenocarcinoma. Moreover, they suggest that inhibition of TGFbetaRI with a small-molecule inhibitor may be effective as a therapeutic approach to treat human pancreatic cancer.

Facultative symbionts are associated with host plant specialization in pea aphid populations.

The pea aphid, Acyrthosiphon pisum, shows significant reproductive isolation and host plant specialization between populations on alfalfa and clover in New York. We examine whether specialization is seen in pea aphids in California, and whether fitness on alternative host plants is associated with the presence of bacterial symbionts. We measured the fitness of alfalfa- and clover-derived aphids on both types of plants and found no evidence for specialization when all aphid lineages were considered simultaneously. We then screened all aphids for the presence of four facultative bacterial symbionts: PAR, PASS, PABS and PAUS. Aphids with PAUS were host-plant specialized, having twice as many offspring as other aphids on clover, and dying on alfalfa. Other aphids showed no evidence of specialization. Additionally, aphids with PABS had 50% more offspring than aphids with PASS when on alfalfa. Thus, specialist and generalist aphid lineages coexist, and specialization is symbiont associated. Further work will resolve whether PAUS is directly responsible for this variation in fitness or whether PAUS is incidentally associated with host-plant specialized aphid lineages.