SUMMARY We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome. This resource provides a powerful tool for biotechnological and eukaryotic cell biology research. Comprehensive gene dispensability comparisons with budding yeast, the first time such studies have been possible between two eukaryotes, revealed that 83% of single copy orthologues in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than non-essential genes to be single copy, broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

Proteolysis of influenza virus hemagglutinin by host cell proteases is essential for viral infectivity, but the proteases responsible are not well defined. Recently, we showed that engineered expression of the type II transmembrane serine proteases (TTSPs) TMPRSS2 and TMPRSS4 allows hemagglutinin (HA) cleavage. Here we analyzed whether TMPRSS2 and TMPRSS4 are expressed in influenza virus target cells and support viral spread in the absence of exogenously added protease (trypsin). We found that transient expression of TMPRSS2 and TMPRSS4 resulted in HA cleavage and trypsin-independent viral spread. Endogenous expression of TMPRSS2 and TMPRSS4 in cell lines correlated with the ability to support the spread of influenza virus in the absence of trypsin, indicating that these proteases might activate influenza virus in naturally permissive cells. Indeed, RNA interference (RNAi)-mediated knockdown of both TMPRSS2 and TMPRSS4 in Caco-2 cells, which released fully infectious virus without trypsin treatment, markedly reduced the spread of influenza virus, demonstrating that these proteases were responsible for efficient proteolytic activation of HA in this cell line. Finally, TMPRSS2 was found to be coexpressed with the major receptor determinant of human influenza viruses, 2,6-linked sialic acids, in human alveolar epithelium, indicating that viral target cells in the human respiratory tract express TMPRSS2. Collectively, our results point toward an important role for TMPRSS2 and possibly TMPRSS4 in influenza virus replication and highlight the former protease as a potential therapeutic target.

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The IOP obtained by the TonoVet(®) and TonoPen XL(®) would be affected by variations in the CCT. Therefore, the CCT should be considered when interpreting IOP values measured by tonometers in dogs.

To understand the regulation of cap-dependent translation initiation mediated by specific 5 untranslated region (UTR) RNA-protein interactions in mammalian cells, we have studied the selective translation of influenza virus mRNAs. Previous work has shown that the host cell mRNA binding protein guanine-rich sequence factor 1 (GRSF-1) bound specifically to conserved viral 5 UTR sequences and stimulated translation of viral 5 UTR-driven mRNAs in vitro. In the present study, we have characterized the functional domains of GRSF-1 and mapped the RNA binding activity of GRSF-1 to RRM 2 (amino acids 194 to 275) with aminoterminal deletion glutathione S-transferase (GST)-GRSF-1 proteins. When these mutants were assayed for functional activity in vitro, deletion of an Ala-rich region (⌬[2-94]) appeared to diminish translational stimulation, while deletion of the Ala-rich region in addition to RRM 1 (⌬[2-194]) resulted in a 4-fold increase in translational activation over wild-type GRSF-1 (an overall 20-fold increase in activity). We have also mapped the GRSF-1 RNA binding site on influenza virus NP and NS1 5 UTRs, which was determined to be the sequence AGGGU. With polysome fractionation and cDNA microarray analysis, we have identified cellular and viral mRNAs containing putative GRSF-1 binding sites that were transcriptionally up-regulated and selectively recruited to polyribosomes following influenza virus infection. Taken together, these studies demonstrate that RRM 2 is critical for GRSF-1 RNA binding and translational activity. Further, our data suggest GRSF-1 functions by selectively recruiting cellular and viral mRNAs containing 5 UTR GRSF-1 binding sites to polyribosomes, which is mediated through interactions with cellular proteins.The ability of cells to respond to extracellular stimuli and intracellular cues, including mitogenic signals, is directly linked to the regulation of mRNA translation initiation. The control of initiation can be regulated by the specific interaction of RNA binding proteins and initiation factors (eIFs) with cisacting elements contained in both the 5Ј and 3Ј untranslated regions (UTRs) of mature mRNAs (reviewed in reference 37). Accordingly, deregulation of protein synthesis is a key mechanism in both malignant transformation and viral replication. Influenza virus infection results in the selective translation of viral mRNAs, while host cell protein synthesis is markedly attenuated (reviewed in reference 38). The subversion of the host cell protein synthetic machinery to produce high levels of influenza virus proteins, which are required for infection and replication, is dependent on conserved sequences present in the 5Ј UTRs of the influenza virus mRNAs (13).Translation initiation relies upon the interactions of transacting factors with both ribosomal RNAs and cis-acting determinants in the mRNA. Influenza virus protein synthesis is a cap-dependent process mediated by highly conserved sequences contained in the 5Ј UTRs of the viral mRNAs (15). As

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