Inhibition of influenza A virus replication by short double-stranded oligodeoxynucleotides.

Influenza A virus causes prevalent respiratory tract infections in humans. Small interfering RNA (siRNA) and antisense oligonucleotides (asODNs) have been used previously for silencing the RNA genome of influenza virus. Here, we explored the use of partially double-stranded oligodeoxynucleotides (dsODNs) to suppress the production of influenza A virus in cell cultures and animal models. We were able to inhibit influenza A virus replication in cultured human lung cells as well as in the lungs of infected C57BL/6 mice by treatment with dsODN 3-h post-infection. In about 20% of the cases (15/77) the titer was reduced by 10- to 100-fold and in 10% up to 1,000-fold. The antiviral effects of dsODNs were dose-dependent, sequence-dependent and comparable to those of its antisense and siRNA analogues. Thus, dsODNs may be developed as an additional class of nucleic acids for the inhibition of influenza virus replication.

AgSwe1p regulates mitosis in response to morphogenesis and nutrients in multinucleated Ashbya gossypii cells.

Nuclei in the filamentous, multinucleated fungus Ashbya gossypii divide asynchronously. We have investigated what internal and external signals spatially direct mitosis within these hyphal cells. Mitoses are most common near cortical septin rings found at growing tips and branchpoints. In septin mutants, mitoses are no longer concentrated at branchpoints, suggesting that the septin rings function to locally promote mitosis near new branches. Similarly, cells lacking AgSwe1p kinase (a Wee1 homologue), AgHsl1p (a Nim1-related kinase), and AgMih1p phosphatase (the Cdc25 homologue that likely counteracts AgSwe1p activity) also have mitoses distributed randomly in the hyphae as opposed to at branchpoints. Surprisingly, however, no phosphorylation of the CDK tyrosine 18 residue, the conserved substrate of Swe1p kinases, was detected in normally growing cells. In contrast, abundant CDK tyrosine phosphorylation was apparent in starving cells, resulting in diminished nuclear density. This starvation-induced CDK phosphorylation is AgSwe1p dependent, and overexpressed AgSwe1p is sufficient to delay nuclei even in rich nutrient conditions. In starving cells lacking septins or AgSwe1p negative regulators, the nuclear density is further diminished compared with wild type. We have generated a model in which AgSwe1p may regulate mitosis in response to cell intrinsic morphogenesis cues and external nutrient availability in multinucleated cells.

A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii.

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Delta deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.