Influenza virus, the causative agent of the common flu, is a worldwide health problem with significant economic consequences. Studies of influenza virus biology have revealed elaborate mechanisms by which the virus interacts with its host cell as it inhibits the synthesis of cellular proteins, evades the innate antiviral response, and facilitates production of viral RNAs and proteins. With the advent of DNA array technology it is now possible to obtain a large-scale view of how viruses alter the environment within the host cell. In this study, the cellular response to influenza virus infection was examined by monitoring the steady-state mRNA levels for over 4,600 cellular genes. Infections with active and inactivated influenza viruses identified changes in cellular gene expression that were dependent on or independent of viral replication, respectively. Viral replication resulted in the downregulation of many cellular mRNAs, and the effect was enhanced with time postinfection. Interestingly, several genes involved in protein synthesis, transcriptional regulation, and cytokine signaling were induced by influenza virus replication, suggesting that some may play essential or accessory roles in the viral life cycle or the host cell's stress response. The gene expression pattern induced by inactivated viruses revealed induction of the cellular metallothionein genes that may represent a protective response to virus-induced oxidative stress. Genome-scale analyses of virus infections will help us to understand the complexities of virus-host interactions and may lead to the discovery of novel drug targets or antiviral therapies.Although it has been nearly 7 decades since the isolation of human influenza virus (34), it remains a world health threat with large economic consequences (28, 43, 52). Although vaccine and drug strategies have managed to contain the spread of the disease and the severity of its symptoms, recent outbreaks, such as the one in Hong Kong in 1997, emphasize the need for continued research efforts for influenza prevention. An abundant but often overlooked source of potential antiviral targets are those cellular genes whose expression is most affected by viral infection. With DNA microarray technology it is now possible to measure the mRNA levels of thousands of cellular genes under a variety of experimental conditions. This approach is increasingly being used to monitor cellular gene expression in response to viral infections (5,19,20,25,30,55,59), expression of viral genes (21, 31, 58), or treatment with antiviral compounds such as interferon (12).Influenza virus is a negative-stranded RNA virus that induces a profound inhibitory effect on the synthesis of cellular proteins. Much of this effect occurs at a posttranscriptional level, as viral RNAs are selectively translated while the initiation and elongation of cellular proteins are inhibited (15). On the other hand, viral proteins carry out a variety of functions within the nucleus, such as removing 5Ј methyl caps from host cell mRNAs (50), blocking ...
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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